Table of Contents

Volume 3, Issue 9

  September 2004

  Pages 796 - 915

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EDITOR'S CORNER

Open Access Article

Cover Image



http://dx.doi.org/10.4161/cbt.3.9.1159
Abstract | PDF

The cover image demonstrates the recruitment of 111In-oxine labeled murine neuronal progenitor cells (C17.2-luc), carrying a luciferase reporter, to subcutaneously implanted Lewis lung carcinomas in Nude mice. C17.2-luc cells were systemically administered via i.v. injection into tumor bearing (lower panels) or control (upper panels) animals, and their accumulation in target sites determined via both nuclear imaging with a Siemens M-Cam and via bioluminescence imaging following luciferin injection. The radioactive decay or luminescence signals were rendered according to the color scale, and overlaid on white light images of the mice to co-register the location of signal origin.




NEWS

Open Access Article

Avoiding Malignancy with Trans-Splicing Gene Therapy


Pages 791 - 796
http://dx.doi.org/10.4161/cbt.3.9.1149
Abstract | PDF

Previous attempts in mice to correct a rare inherited immune disorder, called Hyper IgM X-linked immunodeficiency, have failed because standard gene therapy raised risks for cancer.

Now Weill Cornell Medical College researchers believe they've found a way around that problem.

Reporting in the July 25 issue of Nature Medicine, the investigators used a gene therapy strategy called trans-splicing to successfully correct the disease in mice without increasing malignancy risk. Trans-splicing effectively corrects the mutation at a later step in the genetic process -- at the level of messenger RNA (mRNA).

"The next step is to consider moving it to the clinic. Here at Weill Cornell we have the facility for doing that, and as we develop the safety data, our plans are to move it to humans," said lead researcher Dr. Ronald G. Crystal, Chairman of the Department of Genetic Medicine at Weill Cornell Medical College and Chief of the Division of Pulmonary and Critical Care Medicine at NewYork-Presbyterian Hospital/Weill Cornell Medical Center.

Hyper IgM X-linked immunodeficiency is a rare disease affecting a few hundred children and young adults in the United States. Caused by a genetic breakdown in immune antibody production, individuals with this disease "can't make the right array of antibodies to protect against pathogens in the environment," leaving them vulnerable to lethal infections, Dr. Crystal explained.

Although bone marrow transplants can help prolong life, a majority of patients affected with the disorder die before reaching the age of 30.

Luckily, advances in genomics may someday give these patients a new treatment option.

"In the immune system, immune cells are primarily made in the bone marrow, so if we want to attack this by trying to correct it genetically, we know that's where the cells are that we want to correct," Dr. Crystal explained.

Reduced production of an immune cell protein called CD40 ligand lies at the heart of Hyper IgM X-linked immunodeficiency. Previous studies in mice used standard gene therapy -- where doctors simply introduce a "normal" gene into bone marrow stem cells -- to try and repair the problem.

However, standard gene therapy caused the gene responsible for CD40 ligand production to work overtime, producing excessive amounts of the protein. According to Dr. Crystal, too much CD40 ligand can be as bad as too little, in that it can trigger lymphomas.

"So by putting in the normal gene, but having it turned on all the time, the mice developed cancer," he said.

His team of researchers decided to attack the problem from a different angle. As Dr. Crystal explained, the gene-regulated production of cellular proteins occurs in a step-by-step fashion, with DNA producing a second molecule, called messenger RNA (mRNA), which works to produce cellular proteins like CD40 ligand.

"Unfortunately, individuals with Hyper IgM X-linked immunodeficiency make mRNA that has a mutation in it, so they can't make the CD40 ligand protein," Dr. Crystal said.

With trans-splicing, "we are putting in a gene that makes a piece of correct 'decoy' mRNA that's attractive to the genetic process. This means that the natural mRNA, as it's being processed or 'spliced,' integrates into our decoy instead. In this way it repairs the genetic defect."

Fixing the problem at the mRNA level leaves DNA gene regulation intact, getting around the problem of excessive CD40 ligand production that occurs with standard gene therapy.

The result? In mice bred with X-linked immunodeficiency with Hyper IgM, treatment with this trans-splicing gene therapy "not only repaired the genetic disease, but also avoided cancers," Dr. Crystal said.

Treated mice successfully fended off infection with Pneumocystis carinii, a type of pneumonia that can prove lethal to people with this illness.

Dr. Crystal noted that this gene therapy technique might also be used to treat a wide range of genetic illnesses. "We're simply correcting at the second step, at the level of mRNA," he said.

The next step for his team at Weill Cornell is to test out the technique in humans. According to Dr. Crystal, preparations for clinical trials are already underway.

Study co-researchers include Drs. Minoru Tahara, Robert G. Pergolizzi, and Hiroyasu Kobayashi, of the Weill Cornell Department of Genetic Medicine; Drs. Anja Krause and Karsta Leuttich, of the Weill Cornell Belfer Gene Therapy Core Facility; and Dr. Martin L. Lesser, of the Biostatistics Unit at the North Shore-Long Island Jewish Research Institute, in Manhasset, New York.

This research received funding from the National Heart, Lung, and Blood Institute; the Will Rogers Memorial Fund, Los Angeles; and The Malcolm Hewitt Wiener Foundation, Greenwich, CT.

For more information, please contact: Jonathan Weil; Office of Public Affairs; NewYork-Presbyterian Hospital/Weill Cornell; 525 East 68th Street, Box 144; New York, NY 10021; Tel.: 212.821.0560; Fax: 212.821.0576; Email: jweil@med.cornell.edu

Open Access Article

Polyomavirus BK T-Antigen Protein in Atrophic Precursor Stage of Prostate Cancer


Pages 791 - 796
http://dx.doi.org/10.4161/cbt.3.9.1168
Abstract | PDF

Chances are excellent that your urinary tract is home to a pathogenic organism called the human BK virus. Most of the time, the virus lurks quietly in the kidneys without causing problems. But in people with a depressed immune system - especially those who have just received a kidney transplant - the virus can cause serious kidney and bladder disease.

Now, new research by scientists at the University of Michigan Medical School suggests the intriguing possibility that this common virus also may play a role in prostate cancer - the second-leading cause of deaths from cancer in American men.

A team of scientists directed by Michael J. Imperiale, Ph.D., U-M professor of microbiology and immunology, have found DNA and proteins from the BK virus in prostate tissue with abnormal cell changes. Called atrophic lesions, these changes can be the first step in a series of progressive cell changes leading to prostate cancer.

"Other studies have detected DNA from the BK virus in prostate cancer cells, but this study is the first to pinpoint the location of viral protein expression to one precursor stage in the development of prostate cancer, and to a specific location within prostate cells," Imperiale says. Results from the U-M study, published July 19 in the advance online edition of the journal Oncogene.

"The development of cancer is a multi-step process," Imperiale adds. "Expression of BK viral protein may be just one step among several genetic and environmental factors. We are not saying that BK virus causes prostate cancer, but our results do suggest that the virus plays a role in the transition from normal to uncontrolled growth of prostate cells."

BK is a human virus in the polyomavirus family. It was first discovered in kidney transplant patients who take immunosuppressive drugs to prevent their body from rejecting a new kidney. Scientists have found BK virus in several types of human cancer and it has been shown to cause kidney tumors in laboratory mice. While the virus has received scientific study for its role in kidney disease, only a handful of scientists are studying a possible connection to cancer.

"Polyomaviruses have very small genomes, and they don't have the genes required to copy their own DNA," says Dweepanita Das, Ph.D., a U-M post-doctoral fellow and first author on the paper. "The virus must induce its host cell to divide, so it can use the cell's genes to make proteins the virus needs to reproduce. BK virus uses a protein called T antigen, or TAg, to induce cell division. The problem is that when you interrupt the normal cell cycle to force a cell to divide, sometimes the cell continues to divide abnormally."

U-M scientists analyzed 21 samples of prostate tissue from men with adenocarcinoma whose prostates were surgically removed to prevent the spread of cancer. Tissue samples were obtained from the Tissue Procurement Core at the U-M's Comprehensive Cancer Center. Samples contained benign, pre-cancerous and cancerous tissue.

Using new TAg-specific protein assays they developed, the research team searched the tissue to identify cells containing TAg. Scientists also removed DNA from the prostate tissue samples for sequencing and identification in the U-M's DNA Sequencing Core facility.

The results showed that 71 percent of the prostate samples analyzed for the study contained gene segments from the BK virus. TAg protein from the BK virus was present in 43 percent of the tissue samples, but only in atrophic lesions - a precursor stage of prostate cancer development. TAg was not expressed in normal cells or cancerous cells.

"Atrophic lesions comprise a spectrum of architectural and cytological differences in the appearance of epithelial cells lining the glands in the prostate," says Rajal B. Shah, M.D. M.B.B.S., a clinical assistant professor of pathology and urology in the U-M Medical School and co-author of the study. "Several recent studies have demonstrated that they potentially represent the first stage in a series of progressive cell changes, which can lead to prostate cancer."

In addition to TAg protein, atrophic lesions also contained high levels of protein expressed by the p53 tumor suppressor gene. When U-M scientists discovered that TAg and p53 proteins were located together in the cytoplasm of atrophic lesion cells, but not in the nucleus, an important piece of the puzzle fell into place.

"We know that for p53 to function as a tumor suppressor gene, its protein product must be in the cell's nucleus," explains Imperiale. "TAg apparently sequesters p53 protein in the cell's cytoplasm, preventing it from entering the nucleus and giving the signal for the cell to stop dividing and die."

Imperiale's working hypothesis is that the BK virus infects epithelial cells in the prostate and transforms them into atrophic lesions through TAg expression. This produces an area of uncontrolled cell growth, which - if circumstances are right - can eventually develop into prostate cancer.

If he's right, prostate cancer may have something in common with cervical cancer, which is caused by a different virus - the human papillomavirus.

"Papillomavirus makes proteins with the same function as T antigen," Imperiale says. "They induce cell growth and inactivate the p53 gene, preventing it from killing abnormal cells. So, it's a logical possibility that a similar mechanism occurs in at least some cases of prostate cancer, but much additional research will be needed before we can know for sure."

The U-M study was funded by a grant from the U.S. Army's Medical Research and Material Command, Department of Defense.

For more information contact Sally Pobojewski at pobo@umich.edu or at (734) 615-6912.

Open Access Article

New Gene Signatures Discriminate between Drug-Sensitive and -Resistant ALL


Pages 791 - 796
http://dx.doi.org/10.4161/cbt.3.9.1177
Abstract | PDF

Finding that specific groups of genes are associated with resistance to particular drugs is an important step in identifying new targets for anti-leukemia therapies. A relatively small number of genes are linked to either resistance or sensitivity to four major cancer drugs used to treat acute lymphoblastic leukemia (ALL), suggesting that these genes are key to treatment outcome. This study, by investigators from St. Jude Children's Research Hospital and Erasmus University Medical Center/Sophia Children's Hospital, Rotterdam, The Netherlands, is published in the Aug. 4, 2004 issue of the New England Journal of Medicine (NEJM).

The researchers identified four groups of genes, each of which had a characteristic pattern of expression in leukemia cells depending on whether they were sensitive or resistant to four widely used antileukemic agents. Investigators found that the expression pattern of these genes was significantly related to treatment outcome.

The study identified 123 previously unrecognized genes that are associated with resistance to cancer chemotherapy. Only three of these genes had been previously linked to drug resistance. These new genes represent potential targets for new agents that could be developed to overcome resistance to drugs currently used to treat ALL.

Despite the pioneering role of St. Jude in increasing the cure rate of childhood ALL from 4 percent to more than 80 percent during the past 40 years, the cause of failure in the remaining 20 percent of children is largely unknown, according to William E. Evans, Pharm.D., St. Jude scientific director. The current findings could help further reduce the number of failures. Evans is a senior author of the NEJM report.

"We've known for years that certain genetic changes in leukemic cells are associated with a high risk of treatment failure," Evans said. "But we also realized that many children who have an unfavorable genetic subtype of ALL are cured, and many patients whose genetic subtype was considered to be favorable weren't cured. The findings of this study are helping us understand why patients respond differently to treatment and point to new approaches to overcome these causes of disease relapse."

The researchers tested leukemic cells from 271 children newly diagnosed with ALL for sensitivity to four anti-cancer drugs: prednisolone, vincristine, L-asparaginase and daunorubicin. Using DNA microarray technology, the team determined the levels of expression of thousands of genes in each patient's leukemia cells, and then compared the level of expression (i.e., increased or decreased) of genes in cells that were resistant to each drug to the level of expression of genes in cells that were sensitive to the same drug.

The main study group included 173 children enrolled on the ALL-IX Dutch Childhood Oncology Group protocol at the Erasmus University/Sophia Children's Hospital, or on the German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia 92 and 97 protocols.

The study found a distinct group of genes that had a specific pattern of expression in leukemia cells according to their sensitivity or resistance to each of the four drugs. In addition, the study identified 123 genes linked to drug resistance and showed that the pattern of expression of these genes is significantly related to the risk of disease relapse. The ability of these genes to forecast relapse was then validated in a completely independent group of 98 children who were treated with these same drugs, but on a different treatment protocol at St. Jude.

"The gene expression patterns linked to drug resistance were particularly important since they occurred in both the Rotterdam and the St. Jude patient populations, even though these two groups of children were treated with these drugs in different countries and on different protocols," said Rob Pieters, M.D., chair of Pediatric Oncology/Hematology at Erasmus University, and a senior author of the paper. "This is strong evidence of the link between these resistance genes and treatment outcome."

In addition, the researchers found that there is no universal cross-resistance gene that would block sensitivity to all four drugs. Rather, genes linked to resistance belonged to different functional categories (i.e., they did different tasks in the cell) and these differed for each medication.

"Learning the function of these genes gives us new insights into the causes of resistance to individual drugs used to treat children with ALL," said Monique den Boer, co-author of the study and head of the research laboratory of Pediatric Oncology/Hematology at the Erasmus University.

The study further supports the practice of combination anti-cancer drug therapy to treat ALL.

"The fact that resistance genes fall into groups that perform different functions in the cells demonstrates the importance of using combination therapy to treat cancer," said Ching-Hon Pui, M.D., director of the St. Jude Leukemia/Lymphoma division, the F.M. Kirby Clinical Research Professor for the American Cancer Society and a co-author of the paper.

Combination therapy allows physicians to use multiple anti-cancer drugs to attack different biochemical functions simultaneously.

Other authors of the study include Amy Holleman and Karin M. Kazemier, (Erasmus University Medical Center/Sophia Children's Hospital, Rotterdam, The Netherlands); Meyling H. Cheok, Wenjian Yang, Deqing Pei, Cheng Cheng and Mary V. Relling (St. Jude); Anjo J. P. Veerman (Free University Medical Center, Amsterdam, The Netherlands); and Gritta E. Janka-Schaub (Children's University Hospital, Hamburg, Germany).

This work was supported in part by the National Institutes of Health; the American Cancer Society; ALSAC; the Pediatric Oncology Foundation, Rotterdam; the Nijbakker-Morra Foundation; and the René Vogels stipendium 2002, The Netherlands.

Open Access Article

Cloning of Cancer-Prone Mice from a Melanoma Cell


Pages 791 - 796
http://dx.doi.org/10.4161/cbt.3.9.1178
Abstract | PDF

Some cancer cell changes can be reversed, cloning study finds

Nature can reset the clock in certain types of cancer and reverse some of the elements responsible for causing malignancy, reports a research team from the Whitehead Institute for Biomedical Research and the Dana-Farber Cancer Institute.

The scientists, led by Rudolf Jaenisch, MD, of the Whitehead Institute, and Lynda Chin, MD, of Dana-Farber, demonstrated this phenomenon by cloning normal mice from an advanced mouse melanoma cell.

"This settles a principal biological question," says Jaenisch, a noted cloning expert. "The epigenetic elements of cancer are reversible; the genetic elements, as expected, are not." The work was reported in the August issue of the journal Genes and Development.

Cells fundamentally become cancerous because of mutations, or adverse alterations, in their genes, and these genetic changes cannot be undone. "Epigenetic" factors don't involve alterations in genes, but are changes in gene activity caused by outside biochemical factors, including the effects of regulatory genes.

"One of the important implications of these findings is that it shows the epigenetic alterations in a cancer cell contribute to the malignant characteristics of the cell," says Chin.

Konrad Hochedlinger and Robert Blelloch, postdoctoral researchers in Jaenisch's lab, studied whether any of these epigenetic influences can be reversed. First, they removed the nucleus from a melanoma cell and injected it into a normal, de-nucleated egg cell (a process known as nuclear transfer). The environment of the normal oocyte, or egg cell, "reprogrammed" the cancer cell's genome (complete set of genes in the nucleus) so that it could start over again and redevelop as a normal embryo.

After the altered egg cell had grown to a multicellular blastocyst stage, Hochedlinger and Blelloch harvested embryonic stem cells from it. The stem cells, which are capable of turning into any kind of cell in the body, were then inserted into healthy mouse blastocysts. Many of these blastocysts later developed into normal adult mice.

Nevertheless, when certain cancer-related genes in these mice were activated, they developed malignant tumors at a much faster rate than the control mice.

According to Chin, a dermatologist and cancer researcher, the research opens up the door to developing animal models of cancer enabling researchers to study the relative contributions of the epigenetic versus genetic lesions in the development of cancer.

"The implication is that if you can use drugs to target the epigenome (the non-genetic cancer-causing factors), you can alter the fate of the tumor cell," says Chin. "This may prove to be a key therapeutic opportunity for diverse cancers."

Open Access Article

Innate Glycoprotein Kills H. pylori and May Impact Stomach Cancer


Pages 791 - 796
http://dx.doi.org/10.4161/cbt.3.9.1179
Abstract | PDF

An international team led by The Burnham Institute's Minoru Fukuda, Ph.D., has discovered that a human glycoproteininhibits Helicobacter pylori ("H. pylori"), the bacterium that causes stomach ulcers and is linked with 90% of stomach cancers. Published on August 13th in Science magazine, these results present a new way of looking at treating chronic inflammation associated with stomach ulcers, and introduces the possibility of preventing stomach cancer associated with H. pylori.

Over fifty percent of the world's population is infected with H. pylori, yet only 2% are afflicted with stomach ulcers and only 1% with stomach cancer. A collaboration between The Burnham Institute and Japan's Shinsu University has discovered the defense mechanism that protects the stomach against H. pylori infection.

H. pylori is found mostly in the stomach, where it thrives in the superficial mucin layer lining the stomach. The bacterium is rarely found in the deeper portion of the mucin layer, where the mucous cells produce a particular class of glycoproteins, called O-glycans, linked with the carbohydrate alpha 1,4-N-acetylglusosamine, cloned previously in Dr. Fukuda's laboratory. Because the alpha 1,4-linked N-acetylgucosamine is confined to the stomach's deeper mucosa lining, which also lacks H. pylori, the scientists investigated the possibility that it might play a role against infection by H. pylori.

They isolated mucin from the upper and lower layers and found a key difference: surface-derived mucin actively supported H. pylori growth, while mucins from the second layer inhibited growth. H. pylori in the presence of alpha 1,4-linked N-acetylgucosamine lost its shape, became immobile, and eventually died. This cell-growth immobilizing effect is very similar to the effect of antibiotics, which dissolve or "lyse" the bacterium's cell wall.

The researchers lysed H. pylori cells and set up a biochemical assay using mass spectrometry to analyze the bacterium's cell wall components. They discovered a cholesterol unique to H. pylori, cholesteryl-alpha-D-glucopyranoside, which is a major component of the bacterium's cell wall essential for its growth. Additional experiments confirmed that the O-glycans capped with alpha 1,4-linked N-acetylglucosamine blocked H. pylori's ability to synthesize the cholesterol.

"This is the first time that a glycoprotein has been shown to behave like an antibiotic," says Dr. Minoru Fukuda, who has devoted 20 years of his research career to the study of glycobiology and cancer. "This naturally-occurring cholesterol offers a very specific target for the design of safer drugs that could treat stomach ulcers and, long-term, prevent stomach cancer linked with H. pylori."

Dr. Fukuda believes that it will be possible to breed cows and genetically engineer soy beans that produce milk bearing the inhibitory O-glycans capped with alpha 1,4-linked N-acetylglucosamine.

"This offers an inexpensive way to help people suffering in less developed countries," says Dr. Fukuda. "If we could use transgenic cows and plants to produce this milk, we could possibly eradicate H. pylori infection and eliminate stomach cancer."

The Burnham Institute has applied for patents based on this work.

Dr. Minoru Fukuda is a Professor in the Glycobiology Program of The Burnham Institute's Cancer Center. Other co-authors from The Burnham Institute include Dr. Michiko Fukuda, Professor, and Dr. Motohiro Kobayashi, postdoctoral fellow.

This work was done in collaboration with Dr. Jun Nakayama, corresponding author, and Drs. Masatomo Kawakubo, Yuki Ito, Yukie Okimura, Kyoko Sakura, Susumu Kasama, and Tsutomu Katsuyama of Shinsu University, Japan.

This research was supported by grants from the National Cancer Institute and the Department of Defense.

Open Access Article

Serum Proteomics Yield New Biomarkers for Ovarian Cancer


Pages 791 - 796
http://dx.doi.org/10.4161/cbt.3.9.1184
Abstract | PDF

Johns Hopkins Kimmel Cancer Center researchers have designed a blood test to detect ovarian cancer using three proteins found in common in the blood of women with the disease. Their preliminary studies with the new test suggest a molecular signature exclusive to this deadly cancer, known for its ability to remain undetected and spread quickly.

The Hopkins test, described in the August 15 issue of Cancer Research, identifies the proteins as a truncated form of transthyretin, a fragment of ITIH4 and apolipoprotein A1, teased out with a rigorous evaluation of protein patterns present in blood samples from ovarian cancer patients at several U.S. and international hospitals. Other research groups are evaluating ovarian cancer blood tests that use protein profiles consisting of tens of thousands of unidentified molecules.

“By identifying a select group of biomarkers specific to ovarian cancer, we not only know the proteins we are dealing with, but we can trace them back to alterations in the genetic code of ovarian cancer cells,” says Daniel W. Chan, Ph.D., professor and director of the Biomarker Discovery Center at Johns Hopkins. “We are focusing on the markers for which we have good biological reasoning behind their selection, and hope to expand the panel of markers to catch as many variations in ovarian cancer proteins as possible.”

This research was funded by the National Cancer Institute and Ciphergen Biosystems, which has licensed the test. Chan and his co-workers emphasize that the test will not be commercially available for screening the population at large until completion of further validation studies in larger groups of patients. And even then, Chan notes, it is never going to be possible for a blood test to correctly diagnose 100 percent of cancerous tumors 100 percent of the time. “The goal is to come as close as possible to that by using this test in combination with other available diagnostic tools.” They believe, however, that with some refinements it may already have use for helping determine whether a pelvic mass is ovarian cancer.

In a systematic search to find the most promising blood proteins for their test, the Hopkins scientists conducted a multicenter study and used protein chip technology to screen a total of 195 blood samples from two groups of ovarian cancer patients, healthy people, and patients with benign ovarian tumors. A sophisticated bioinformatics program was used to select proteins present at unusually high or low levels in ovarian cancer samples as compared with normal or benign. Samples in the two groups were analyzed separately to account for differences in patient populations and sample collection techniques. Then, researchers compared protein profile results in these two groups and ultimately narrowed the search for potential marker candidates to the three proteins, one of which (ITIH4) is commonly found at high levels in ovarian cancer and the other two at lower levels.

“Typically, only half of early-stage ovarian cancer patients have elevated blood levels of a standard marker called CA125,” says Zhen Zhang, Ph.D., associate professor and associate director of the Biomarker Discovery Center at Johns Hopkins. “By combining our discovered proteins into a multi-marker assay, it may improve detection of stage I/II ovarian cancer.” The new proteins were screened against a separate collection of blood samples from patients with normal and cancerous tissues. Of 23 patients with Stage I/II invasive ovarian cancer, the three protein markers alone and combine with CA125 correctly identified cancer 74 percent of the time (17 of 23) as compared to 65 percent (15 of 23) with CA125 alone. Although the sample size was too small for this difference to be statistically significant, the scientists conducted further studies lowering the cutoff value for CA125 to below current standards. The new test plus CA125 as well as CA125 alone detected 83 percent (19 of 23) of the cancers. In addition, the new test plus CA125 correctly identified healthy samples 94 percent of the time (59 of 63) as compared to 52 percent (33 of 63) for CA125 alone.

To verify that the candidate markers were specific to ovarian cancer, the scientists also compared results of the protein profiles with a separate group of blood samples from 142 Johns Hopkins ovarian, breast, colon, prostate cancer patients and healthy people. Protein markers from Hopkins’ ovarian cancer samples matched those from the other two groups of blood samples. Breast, colon and prostate cancer samples exhibited levels of the three proteins closer to those of normal patients, indicating that the markers may be exclusive to ovarian cancer.

The scientists will conduct further studies to map all three proteins to the genetic pathways linked to ovarian cancer development and combine the blood test with radiologic tools such as ultrasound. They also will search for more proteins to add to the current panel of markers.

Additional research participants included Robert Bast Jr. and Yinhua Yu from the M.D. Anderson Cancer Center; Jinong Li, Lori Sokoll, Alex Rai, Jason Rosenzweig, Bonnie Cameron, and Young Wang from Johns Hopkins; Andrew Berchuck from Duke University Medical Center; Carolien van Haaften-Day and Neville Hacker from The Royal Hospital for Women, Australia; Henk de Bruij and Ate van der Zee from University Hospital Groningen, the Netherlands; Ian Jacobs from Bart’s and The London, Queen Mary’s School of Medicine, United Kingdom, and Eric Fung from Ciphergen Biosystems.

Zhang, Z. et al, “Three Biomarkers Identified from Serum Proteomic Analysis for the Detection of Early Stage Ovarian Cancer,” Cancer Research 2004; 64.

Under a licensing agreement between Ciphergen Biosystems, Inc. and the Johns Hopkins University, Chan is entitled to a share of royalty received by the University on sales of products described in this article. Chan is entitled to Ciphergen Biosystems, Inc. stock options, which is subject to certain restrictions under University policy. Chan also is a consultant to Ciphergen Biosystems, Inc. and a member of the company’s scientific advisory board. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

Open Access Article

Circulating Tumor Cells in Aggressive Breast Cancer


Pages 791 - 796
http://dx.doi.org/10.4161/cbt.3.9.1188
Abstract | PDF

Patients with advanced breast cancer who have more than five circulating tumor cells in the blood may have a more dangerous form of the disease, according to a study published in the Aug. 19 issue of The New England Journal of Medicine.

The pivotal study could lead to more tailored treatments that would spare some women from the most potent chemotherapy, or, conversely, recognize which patients need more aggressive therapy at the start of treatment, says the study's lead author Massimo Cristofanilli, M.D., associate professor in The University of Texas M. D. Anderson Cancer Center's Department of Breast Medical Oncology.

"This is the first time that we can actually stratify metastatic breast cancer patients based on their risk," says Cristofanilli. "When a physician assesses a woman with metastatic breast cancer, it is very difficult to make an accurate prediction of her prognosis. Now we may know more about what the prognosis will be, based on a simple blood test and a new technology. One day we may be able to suggest to a patient - based on personal risk - a more aggressive treatment, a less aggressive treatment, or no treatment at all."

çMetastasis is the most life-threatening aspect of cancer, says Cristofanilli. To metastasize, cancer cells must leave the site of the primary tumor, travel through the blood and proliferate in a new site. Until recently, doctors have not been able to reliably isolate circulating tumor cells in the blood. Within the last few years, several methods have been developed to label tumor cells with antibodies that can then be measured precisely, identifying even one tumor cell in a vial of blood.

The prospective multi-center trial was conducted at 20 institutions in the United States - including the Cleveland Clinic, Duke University, the University of Arizona, and the University of Michigan - with M. D. Anderson as the lead site. In total, 177 women with metastatic disease were enrolled either prior to starting initial therapy or when changing to a new course of treatment. Patients were first tested for circulating tumor cell counts prior to therapy, and then again at first follow-up approximately three to four weeks later.

In the first test, 49 percent, or 87 women, were found to have five or more circulating tumor cells per 7.5 milliliters of blood, the equivalent of one blood draw. These patients had significantly shorter progression-free survival (2.7 months versus seven months) and overall survival (10.1 months versus greater than 18 months) than women with fewer than five circulating tumor cells per blood draw.

At the follow-up visit, circulating tumor cells again were collected. Then, 30 percent of the women were found to have five or more circulating tumor cells per blood draw, indicating a portion of the study group responded to therapy. The difference in progression-free survival between the two groups remained consistent - 2.1 months for women with five or more circulating tumor cells versus seven months for women with less than five circulating tumor cells. Overall, survival in the women with more than five circulating tumor cells was 8.2 months, compared to greater than 18 months in the cohort with less than five circulating tumor cells.

Moreover, says Cristofanilli, the presence of cancer cells in the blood predicted prognosis more accurately than the site of metastatic disease or the presence of estrogen receptor on the tumor cells, thereby having even more potential to impact standard treatment and future research.

"You can see there is a difference in efficacy or benefit of treatment in women who are selected based on the presence of cells or not," says Cristofanilli. "The most obvious case is estrogen-receptor positive disease. Some doctors are reluctant to give these women a hormonal treatment at diagnosis, but would rather be safe and give chemotherapy, the most aggressive treatment. Utilizing this test, we may one day definitively tell estrogen-receptor positive women if they have a worse prognosis and that chemotherapy is the right approach. Or, for those with few or no circulating cells, it is safe to go ahead with hormonal treatment alone."

Cristofanilli and his M. D. Anderson colleagues have long been working with circulating tumor cell technology and were the first to recognize the potential prognostic implications of its detection in women with metastatic disease. At the 2003 annual American Association of Cancer Research meeting, they reported a preliminary analysis of 41 patients evaluated at M. D. Anderson. These initial observations are further validated by the report of the results of this multi-center trial.

Cristofanilli would like to see circulating tumor status used prospectively to group patients for future clinical trials. Other future plans, says Cristofanilli, are to collect the circulating tumor cells, look at gene expression and see how it is representative of the primary tumor - perhaps, then doing away the need for a biopsy in the metastatic setting with the ability to evaluate how the patient is responding to therapy. In addition, he would like to study the correlation of circulating tumor cells to how patients respond to various types with treatments.

"If you look carefully, patients who are considered to have similar disease by standard clinical criteria may in fact have tumors that have quite different biological characteristics," he says. "Some patients might do better no matter what they receive."

The diagnostic technology utilized in this study, the CellSearch™ System, is marketed by Veridex, LLC, a Johnson & Johnson company. It is the first of its kind to automate the detection and enumeration of circulating tumor cells in peripheral blood, and works by detecting cancer cells that detach from solid tumors and enter the blood stream. The test is not yet commercially available to patients; it is expected to be available in fall 2004.

Cristofanilli cautions that the information generated by the technology is a powerful tool for both patient and physicians and must be utilized with caution.

The majority of women facing metastatic breast cancer want to know their prognosis - good or bad - but they are afraid of bad news," he says. "If we can discover in a newly diagnosed patient that tumor cells are already in the blood, both patient and physician would be aware that we are dealing with a more aggressive cancer that requires more aggressive treatment early on." The study was funded by Immunicon Corp. of Huntingdon Valley, Pa.

For more in formation please contact: Julie Penne; Director of Media Communications; Email: jpenne@mdanderson.org

Open Access Article

PTEN Key to Herceptin Response in Her2-Positive Breast Cancer


Pages 791 - 796
http://dx.doi.org/10.4161/cbt.3.9.1191
Abstract | PDF

For many patients with advanced breast cancer, the cancer drug Herceptin (trastuzumab) has offered new hope when traditional cancer drugs failed to work, shrinking tumors and sending some patients into remission.

Now Dihua Yu, M.D., Ph.D., and her colleagues at The University of Texas M. D. Anderson Cancer Center have uncovered a powerful new cancer-fighting property of Herceptin, an antibody-based drug that targets a protein on breast cancer cells called HER-2 (also called ErbB2). The discovery explains why some HER-2 positive patients don't respond as well to the drug and also offers a potential solution that could allow more HER-2 positive patients to benefit from the treatment.

The study, which appears in the August 2004 issue of the journal Cancer Cell, demonstrates that the presence of a protein called PTEN in HER-2 positive patients' tumor cells is a powerful predictor of who will respond to Herceptin. In normal cells, the PTEN protein helps control cell division, but in about half of breast tumors PTEN levels are very low or the protein is completely missing. Those PTEN-missing tumors did not respond to Herceptin treatment.

"Our goal is to allow doctors to quickly and accurately tailor cancer treatment to each individual patient," says Yu, professor of surgical oncology, and the study's principal investigator. "Tailored treatment means giving each patient the medication most likely to benefit her, while simultaneously minimizing side effects."

In a recent clinical trial at M. D. Anderson, 65 percent of HER-2 positive patients taking Herceptin in addition to chemotherapy had a complete response rate, compared to 26 percent taking chemotherapy alone. But doctors have had no way to predict who among HER-2-positive patients, which account for about one-third of all breast cancer patients, is most likely to benefit.

"Previously, it was known that Herceptin binds to the HER-2 protein and causes it to degrade," says Yu. "But this process takes days. What we found is that very quickly, within ten minutes of administration, Herceptin activates PTEN, a powerful tumor suppressor gene. We are adding a very new understanding of how Herceptin works."

The scientists studied the tumors of 47 metastatic HER-2 positive breast cancer patients who had received Herceptin and chemotherapy as well as 37 patients who received chemotherapy alone. PTEN levels varied widely among both groups, but only 11 percent of patients who had a very low level of PTEN responded to Herceptin, versus 66 percent of those with high levels of PTEN. There was no correlation between PTEN level and response to traditional chemotherapy agents called taxanes.

"Our results show PTEN is a very powerful predictor of who will respond to Herceptin," says Yu.

Breast cancer patients whose tumors make too much of the HER-2 protein are at much greater risk of metastasis because the HER-2 protein stimulates cells to grow and spread aggressively. When Herceptin attaches to the HER-2 protein, it interrupts those growth signals, which are sent through a series of signaling proteins inside the cell. PTEN acts as a natural brake on tumor growth by, among other things, blocking the effect of a growth promoting protein called PI3K.

Yu and her colleagues discovered that if they administered a drug that turns off PI3K in breast cancer cells in the laboratory, they became much more sensitive to the effects of Herceptin. The PI3K inhibitor mimicked the action of PTEN and restored the ability of Herceptin to slow or stop the growth of cancer cells.

Yu pointed out that there are several experimental PI3K inhibitors currently in clinical trials for treatment of breast cancer that, if they prove to be safe, could potentially be combined with Herceptin to boost its effectiveness in PTEN-missing breast cancers.

"In the past we looked for patients' HER-2 expression, and if HER-2 was high we gave them Herceptin," says Yu. "In the future, when patients come to M. D. Anderson we will look for patients with high levels of HER-2 and PTEN expression and these are the patients that we expect will benefit most from Herceptin. For those with low PTEN, we hope to be able to offer combination therapy with a PI3K inhibitor that might work synergistically to boost the effectiveness of Herceptin."

Yu says she and her colleagues in the Breast Medical Oncology Department are planning a clinical trial to be conducted at M. D. Anderson in which they would test each patient for HER-2 and PTEN proteins and offer targeted treatment based on the level of each protein.

"There has been a need to develop new markers that can improve the efficacy of Herceptin," says Francisco Esteva, M.D., Ph.D., an associate professor in the Department of Breast Medical Oncology. "This is one of the first studies that allows us to understand the mechanism of Herceptin resistance and offers a potential way to counteract it."

"In the past, selecting a particular chemotherapy drug has been a lottery," says Yu. "Patients had no way of knowing if they would benefit. The new generation of cancer drugs is providing targeted treatment with fewer side effects and giving patients a better chance that a particular treatment will work for them."

The study was funded by grants from the National Institutes of Health and M. D. Anderson Cancer Center. Yu's co-authors include Yoichi Nagata, M.D., Ph.D.; Keng-Hsueh Lan, M.D.; Xiaoyan Zhou; Ming Tan, M.D., Ph.D.; Esteva; Aysegul A. Sahin, M.D.; Kristine S. Klos, Ph.D.; Ping Li; Nina T. Nguyen; Gabriel N. Hortobagyi, M.D. and Mien-Chie Hung, Ph.D. of M. D. Anderson; and Brett P. Monia of ISIS Pharmaceuticals.

Contact: Nancy Jensen, (713) 792-0655, nwjensen@mdanderson.org; or Stephanie Dedeaux, (713) 563-0000, srdedeau@mdanderson.org




REVIEWS

Open Access Article

Shifting paradigms in hormonal therapy for breast cancer

Generosa Grana
Pages 797 - 805
http://dx.doi.org/10.4161/cbt.3.9.1128
Abstract | PDF

Background: Tamoxifen has been the mainstay of endocrine treatment for postmenopausal patients with early and advanced breast cancer for many years. However, as an anti-estrogen with partial estrogenic effects, tamoxifen also has some serious safety and tolerability issues. Following tamoxifen treatment failure there were initially limited options for subsequent therapy. Therefore, new agents, including new anti-estrogens and aromatase inhibitors (AIs) were developed.

Methods: Data for this review were identified by searches of PubMed and references from relevant articles. Abstracts were included only when the relevant information had not been published in full elsewhere. Only papers published in English were included.

Results: The AIs have been shown to be effective treatments for hormone-sensitive metastatic breast cancer.

Conclusions: Current clinical trial data in early breast cancer show longer disease-free survival and time to recurrence with anastrozole than that demonstrated with tamoxifen and very good general tolerability, although longer follow-up is warranted. Clinical trials of the use of AIs in breast cancer prevention and preliminary data for the combination of anastrozole with a gonadotrophin-releasing hormone for the treatment of premenopausal women with early stage breast cancer are also discussed here.

Open Access Article

Perspectives of SEREX-defined antigens in diagnosis and immunotherapy for gastric cancer

Yunwei Wang, Qinlong Gu, Bingya Liu and Zhenggang Zhu
Pages 806 - 811
http://dx.doi.org/10.4161/cbt.3.9.1140
Abstract | PDF

Gastric cancer is the most common malignancy in the world. Often diagnosed at advanced and unresectable stage, there is no curative treatment available for gastric cancer. Immunotherapy might be a promising alternative option. The key challenge in gastric cancer diagnosis and immunotherapy is to find an efficient target that not only provide early indication of the disease but also can upregulate the anti-tumor immune response in the cancer patients. Tumor-associated antigens(TAAs) expressed in tumor cells can elicit the immune response in autologous host and are related to changes during the disease process may fulfill this demanding. Now, Thousands of antigens have been discovered by a powerful method called SEREX(serological analysis of tumor antigen by recombinant cDNA expression libraries) and some of SEREX-defined antigens have been evaluated in clinical trails as vaccines for cancer immunotherapy. This review will provide information on the application of SEREX method in gastric cancer research and knowledge learned from these studies to develop antigen-specific diagnosis and therapy for gastric cancer.




EXEGESES AND VIEWS

Open Access Article

Hydrodynamic consequences of glycolysis: Thermodynamic basis and clinical relevance

Hans Peter Rutz
Pages 812 - 815
http://dx.doi.org/10.4161/cbt.3.9.1134
Abstract | PDF

Since Warburg’s original description of aerobic and anaerobic glycolysis in 1924, the biochemical and molecular bases of glucose metabolism in cells have been characterized. However, its thermodynamic implications have found little attention. Van’t Hoff’s law of thermodynamics was applied here to describe how glucose metabolism affects intracellular osmotic pressure, hence revealing how glucose metabolism provides cells with osmotic energy as well as chemical energy. Ensuing osmosis-driven movement of free water across membranes affects cell volumes and shape, hydration of intracellular components, and interactions of cells with their environment. Comparative analysis of extant data revealed strong evidence for clinically relevant glycolysis-driven effects under hypoxic conditions known to occur in cancer, vascular disease as well as intensive care, including critical and preventable ones affecting treatment effectiveness and thus, clinical outcome. These findings provide the first evidence confirming the existence of glycolysis-driven hydrodynamic effects and suggest that high levels of aerobic and anaerobic glycolysis provide a common biophysical basis in seemingly unrelated medical conditions.




FOCUS ON THE LIVER

Open Access Article

Introduction: Understanding the consequences of epigenetic mechanisms and its effects on transcription in health and disease

Assam El-Osta
Pages 816 - 818
http://dx.doi.org/10.4161/cbt.3.9.1100
Abstract | PDF

Over the course of thirty-five years of research into the chromosome we are confronted with a number of general conclusions central to chromatin biology, perhaps most pertinent of these is that organization of higher order nucleosome structures play a fundamental role in DNA behavior. Folding and packaging of DNA is linked with a number of processes ranging from transcriptional regulation, genome stability, chromosome segregation, including replication and repair. Intrinsic to nuclear function is chromatin structure and the relationship with epigenetic modification. While we are aware that genetic and epigenetic changes can influence tumorigenesis, it is still unclear what protein components are specified on chromatin or the molecular events that underlie the link between chromosomes and cancer. In recent years, our understanding of DNA methylation with cancer development has revealed some surprising findings with particular emphasis on the antithetical character of hypo- and hyper- methylation of DNA. The molecular mechanism by which methylation is responsible for increased cancer risk has received widespread attention and is relavent to our understanding of genetic stability. The biological significance of other epigenetic modifications such as histone tail changes and active chromatin remodeling are also a major driving force in chromosome research in cancer. In light of recent advances in the understanding of epigenetic changes in disease we have organized a focused review series and discuss the significance of Epigenetics in Normal Control and Deregulation in Cancer. Authors from divergent fields of study have contributed articles that examine chromatin dynamics in cancer, bringing together research interests from a wide variety of fields. In this vein, we discuss the consequences of deregulating epigenetic mechanisms with a viewpoint on transcriptional control, chromatin function and human disease.

Open Access Article

MDR1, Chemotherapy and chromatin remodeling

Emma K. Baker and Assam El-Osta
Pages 819 - 824
http://dx.doi.org/10.4161/cbt.3.9.1101
Abstract | PDF

The development of multidrug resistance (MDR) in cancer can severely impede the efficacy of chemotherapy treatment. P-glycoprotein (Pgp) overexpression, encoded by the MDR1 gene, is a well-established mediator of MDR. MDR1 expression is rapidly upregulated by chemotherapeutic drugs and a number of other exogenous stimuli, however the mechanisms underlying its transcriptional regulation remain unclear. In recent years, research has indicated that chromatin accessibility, or epigenetic modifications, will play a large role in controlling the endogenous MDR1 expression state, and its response to activation stimuli. This review examines some of these studies, and discusses how new developments from the greatly expanding epigenetics field may extend to MDR1 transcriptional research.

Open Access Article

Chromatin dynamics and cancer

Valentina Guasconi and Slimane Ait-Si-Ali
Pages 825 - 830
http://dx.doi.org/10.4161/cbt.3.9.1102
Abstract | PDF

Chomatin remodelling multi-protein complexes play a key regulatory role in many physiological processes such proliferation and differentiation. The alteration in the recruitment or in the function of one of these chromatin remodelling factors can lead to a proliferation defect, that might result from the aberrant activation or repression of key genes that regulate cell proliferation. In fact, aberrant chromatin remodelling leads to severe human diseases such as leukaemia, epithelial cancers and Rubinstein-Taybi syndrome.

Open Access Article

Altered epigenetic signals in human disease

Luciano Di Croce, Marcus Buschbeck, A. Gutierrez, I. Joval, L. Morey, R. Villa and S. Minucci
Pages 831 - 837
http://dx.doi.org/10.4161/cbt.3.9.1103
Abstract | PDF

The genetic information of almost all eukaryotic cells is stored in chromatin. In cancer cells, alterations in chromatin organization or in its epigenetic marks occur frequently. Among these are changes in the patterns of DNA and histone methylation. Using Acute Promyelocytic Leukemia as model system we could demonstrate a direct correlation of epigenetic events induced by the driving oncogene product PML-RAR? and cancer progression. Several of the enzymes ultimately responsible for these events can be inhibited by small compound inhibitors and thus can serve as targets in cancer therapy. In this article, we review the role of DNA methylation, histone methylation and chromatin alterations in human diseases. A picture is emerging in which these epigenetic signals “cross-talk” and are implicated in the physiological and pathological spreading of gene silencing.




RESEARCH PAPER

Open Access Article

Murine neuronal progenitor cells are preferentially recruited to tumor vasculature via a4-integrin and SDF-1a dependent mechanisms

Jennifer R. Allport, Vivek R. Shinde Patil and Ralph Weissleder
Pages 838 - 844
http://dx.doi.org/10.4161/cbt.3.9.1036
Abstract | PDF

Recent studies have described neuronal progenitor cell recruitment to tumors in vivo, however, the mechanisms mediating this recruitment are not yet understood. When C17.2 murine neuronal progenitors stably expressing luciferase (C17.2-luc) were adoptively transferred into mice carrying subcutaneous Lewis lung carcinomas they accumulated at 1% injected dose/g of tumor tissue. C17.2-luc demonstrated significantly greater accumulation and transmigration on tumor-derived endothelium (TEC) than on normal endothelium under physiologically relevant flow conditions. Function blocking of a4-integrin reduced recruitment of C17.2-luc cells to normal endothelium but not to TEC, however, function blocking of SDF-1a reduced overall accumulation of C17.2-luc on TEC and specifically reduced transendothelial migration. Together, these data suggest that recruitment of C17.2-luc cells to TEC is mediated via SDF- 1a/CXCR4 activation that results in modification of a4-integrin and results in improved recruitment of C17.2-luc cells.




COMMENTARY

Open Access Article

Could Labeled Neuronal Progenitor Cell Be Used to Target Glioma Tumor Endothelium

Constance Y. Fears, Harald W. Sontheimer, Daniel C. Bullard and Candece L. Gladson
Pages 845 - 846
http://dx.doi.org/10.4161/cbt.3.9.1123
Abstract | PDF

Murine Neuronal Progenitor Cells are Preferentially Recruited to Tumor Vasculature via a4-Integrin and SDF-1a Dependent Mechanisms

Jennifer R Allport, Vivek R Shinde Patil and Ralph Weissleder




RESEARCH PAPER

Open Access Article

Eicosanoid metabolism in squamous cell carcinoma cell lines derived from primary and metastatic head and neck cancer and its modulation by celecoxib

Claudia P. Schroeder, Peiying Yang, Robert A. Newman and Reuben Lotan
Pages 847 - 852
http://dx.doi.org/10.4161/cbt.3.9.1037
Abstract | PDF

Eicosanoid metabolism through cyclooxygenases (COXs) and lipoxygenases (LOXs) generates various lipids that play a role in squamous cell carcinogenesis. We used pairs of head and neck squamous cell carcinoma (HNSCC) cell lines derived from primary and metastatic tumors of the same patient to analyze eicosanoid metabolites by ESILC/ MS/MS and COX/LOX expression by western immunoblotting. The effects of celecoxib on eicosanoid synthesis and HNSCC cell growth were examined. Prostaglandin E2 (PGE2) was the major metabolite in three of six cell lines. COX-2 was detected in three cell lines, which produced PGE2 (two from metastases). We found low expression of COX-1 at similar intensities for each pair of cell lines. 5-LOX was detected in all cells. Some expressed 12-LOX, 15-LOX-1, and 15-LOX-2, but there was no correlation between enzyme expression and endogenous product content. Exogenous arachidonic acid did not change the profile of eicosanoid biosynthesis. Low doses of celecoxib inhibited formation of PGE2 in UMSCC-14A cells by 84% as early as 6 hours. In contrast, 5-HETE, 12-HETE, and 15-HETE levels were increased by approximately 40-, 5-, and 3-fold, respectively, with a decline to baseline levels within 24 hours. High dose celecoxib increased the 12-HETE level 2.3-fold after 3 days of incubation. Celecoxib inhibited growth of all HNSCC cell lines in a concentration-dependent manner regardless of their COX expression (IC50 values after 3 days; 33 to 62 µM). Our findings provide new informations about individual eicosanoids produced by HNSCC cells and their differential regulation by the selective COX-2 inhibitor celecoxib.




COMMENTARY

Open Access Article

Do elevated levels of eicosanoids play a role in head and neck cancer recurrence and metastasis? Implications for prevention and treatment

Durga P. Cherukuri and Mark A. Nelson
Pages 853 - 854
http://dx.doi.org/10.4161/cbt.3.9.1124
Abstract | PDF

Commentary to:



Eicosanoid Metabolism in Squamous Cell Carcinoma Cell Lines derived from Primary and Metastatic Head and Neck Cancer and its Modulation by Celecoxib

Claudia P. Schroeder, Peiying Yang, Robert A. Newman and Reuben Lotan




RESEARCH PAPERS

Open Access Article

Aqueous extract of urtica dioica makes significant inhibition on adenosine deaminase activity in prostate tissue from patients with prostate cancer

Ilker Durak, Hasan Biri, Erdinç Devrim, Sinan Sözen and Aslıhan Avcı
Pages 855 - 857
http://dx.doi.org/10.4161/cbt.3.9.1038
Abstract | PDF

Aim: Investigation of possible effects of aqueous extract of urtica dioica leaves on adenosine deaminase activity in prostate tissue from patients with prostate cancer. Methods: Ten prostate tissues from patients with pathologically proven localized prostate cancer (Gleason scores 4 to 7) were used in the study. In the tissues, ADA activities with and without preincubation with different amounts of urtica dioica extracts were performed. Results: Aqueous extract of urtica dioica results in significant inhibition on adenosine deaminase (ADA) activity of prostate tissue. Conclusion: ADA inhibition by urtica dioica extract might be one of the mechanisms in the observed beneficial effect of urtica dioica in prostate cancer.

Open Access Article

Comparison of genetic changes between interphase and metaphase nuclei in monitoring CML and APL treatment using DC-FISH technique

Tahsin Yakut, Rıdvan Ali, Unal Egeli, Fahir Ozkalemkas, Ilker Ercan, Tülay Özçelik, Vildan Ozkocaman, Barboros Yigit and Ahmet Tunali
Pages 858 - 863
http://dx.doi.org/10.4161/cbt.3.9.1039
Abstract | PDF

In leukemias, the monitoring techniques on the response after the treatment have clinical importance for evaluating new therapeutic approaches and identifying the risk of relapse. In this study, genetic changes before and after chemotherapy in interphase and metaphase nuclei of bone morrow of adults with provisional diagnosis of leukemia were compared to understand the molecular characterization and pathogenesis of the leukemia for the classification of diagnosis and prognosis. We examined bone morrow cells of 47 chronic myeloid leukemia (CML) cases (29 of 47 at the time of diagnosis, 31 of 47 after chemotherapy) with the bcr/abl translocation probes and of 10 acute promyelocytic leukemia (APL) cases (7 of 10 at the time of diagnosis, 4 of 10 after chemotherapy) with the PML/RAR_ translocation probes by using dual color-flourescence in situ hybridization (DC-FISH). For each case, 400 interphase nuclei and 11 to 25 metaphases nuclei were analysed. The ratios of translocations before and after chemotherapy were compared between interphase and metaphase nuclei. After chemotherapy, though, translocations were detected in interphase nuclei of 29 of the 31 CML and 4 of the 4 APL cases, these translocations were determined in metaphase nuclei of only 14 of the 31 CML and 1 of the 4 APL cases with very low ratios (p<0.01). The results showed that the rates of translocation positive interphase nuclei were higher than the rates of translocation positive metaphase nuclei (p<0.01) after chemotherapy, so there may be some factors effecting proliferative activity of metaphase formation in leukemias.

Open Access Article

2-deoxy-D-glucose enhances the cytotoxicity of topoisomerase inhibitors in human tumor cell lines

B. Dwarakanath, Divya Khaitan and T. Ravindranath
Pages 864 - 870
http://dx.doi.org/10.4161/cbt.3.9.1040
Abstract | PDF

Resistance of tumors due to restricted drug accumulation and reversal of DNA lesions in tumor cells as well as normal tissue toxicity limit the efficacy of topoisomerase inhibition based anticancer drugs. It has been proposed that selective inhibition of energy dependent repair processes and enhanced retention of drug in cancer cells can significantly improve the therapeutic efficacy. The purpose of these studies was to verify this proposition by investigating the effects of 2-deoxy-D-glucose (2-DG) an inhibitor of the glycolytic ATP production on the cytotoxicity of certain topoisomerase inhibitors in human tumor cell lines. Human glioma (BMG-1 and U-87) and squamous carcinoma (4197 and 4451) cell lines were investigated with topo-poisons like etoposide (topo II inhibitor), camptothecin (topo I inhibitor) and hoechst-33342 (topo I and II inhibitor). DNA damage induction (halo assay), cell survival (macro colony assay), cytogenetic damage (micronuclei) and apoptosis (morphological analysis) were investigated. Presence of 2- DG for 2 h following exposure to the topoisomerase inhibitors enhanced the cell death (macro colony assay) in a concentration dependent manner and a 2-3-fold increase was observed at 5mM (equimolar with glucose). Halo assay revealed that 2-DG inhibited the reversal of cleavable complex leading to the accumulation of DNA strand breaks. Under these conditions 2-DG enhanced the drug-induced micronuclei formation by nearly2 folds with etoposide and hoechst- 33342 and a 4-fold increase in delayed apoptosis was observed in case of etoposide. These results clearly demonstrate that presence of 2-DG for a few hours following exposure to topo-inhibitors enhances the cytotoxicity, primarily by increasing the cytogenetic damage.

Open Access Article

P53-dependent activation of a molecular beacon in tumor cells following exposure to doxorubicin chemotherapy

Rishita Shah and Wafik S. El-Deiry
Pages 871 - 875
http://dx.doi.org/10.4161/cbt.3.9.1053
Abstract | PDF

In an effort to begin developing a non-invasive strategy for in-vivo detection of the cellular DNA damage response, we engineered a molecular beacon to detect expression of p21(WAF1/CIP1), a gene whose transcription is directly activated by the p53 tumor suppressor protein. Introduction of a phosphorothioate-modified p21-beacon by transfection in human tumor cells led to a slight background signal that increased in a dose dependent manner between 50 and 400 nM beacon. Strong nuclear signal was observed following treatment of wild-type p53-expressing human H460 lung cancer cells for 8 hours with the chemotherapeutic agent doxorubicin (adriamycin). Similar induction was observed in wild-type p53-expressing HCT116 cells. Interestingly, following doxorubicin exposure, there was activation of the p21-beacon in p21-null HCT116 cells, which was not observed in p53-null HCT116, or mutant p53-expressing DLD1 cells that are either wild-type or p21-null. Increased signal from the phosphorothioate-modified p21-beacon in doxorubicin-treated cells likely resulted from sequence-specific hybridization as well as sequence-independent cleavage that may occur due to p53-dependent activation of endonucleases during apoptosis. We conclude that activation of p53 by chemotherapy leads to a strong signal from a p21-beacon that may be useful in further testing both in vitro and in vivo. Strategies need to be developed to optimize the gene or damage specificity as well as the sensitivity to therapeutic response of this non-invasive imaging approach.

Open Access Article

Vascular endothelial growth factor augments human herpesvirus-8 (HHV-8/KSHV) infection

Patrick W. Ford, Khalief E. Hamden, Audy G. Whitman, James A. McCubrey and Shaw M. Akula
Pages 876 - 881
http://dx.doi.org/10.4161/cbt.3.9.1054
Abstract | PDF

Human herpesvirus-8 (HHV-8/KSHV) is etiologically associated with Kaposi’s sarcoma (KS) and other tumors. The Raf oncoprotein enhances HHV-8 infection of target cells. In addition, we have previously demonstrated that Raf induces vascular endothelial growth factor (VEGF) expression. VEGF is a growth factor that has autocrine growth activity and has been implicated in the formation of the spindle shape cell morphology characteristic of Kaposi’s sarcoma (KS). The aim of this study was to test the hypothesis that VEGF enhances infection of HHV-8. Herein, we demonstrate that the soluble VEGF enhanced green florescence protein encoding (GFP)-HHV-8 (rKSHV.152) infection of human foreskin fibroblasts (HFF) and not of 293 cells. We found this to be in part, due to the fact that HFF inherently produces significantly lower concentrations of VEGF when compared to 293 cells. Treating 293 cells (but not HFF) with a VEGF receptor (VEGFR) inhibitor significantly lowered infection. Furthermore, transfecting 293 cells with VEGF specific si-RNA did not alter the binding of HHV-8 to cells; but significantly lowered VEGF expression and thus GFP-HHV-8 infection. Interestingly, lowering VEGF expression in 293 cells wtih VEGF specific si-RNA did not completely inhibit GFP-HHV-8 infection. We conclude that VEGF is not a requirement for HHV-8 infection; but VEGF plays a major role in augmenting infection at a post binding stage of entry. These findings suggest that targeting VEGF/VEGFR may prove efficacious in controlling HHV-8 associated pathogenesis.

Open Access Article

Effects of resveratrol on gene expression in renal cell carcinoma

Ting Shi, Louis S. Liou, Provash Sadhukhan, Zhong-Hui Duan, Andrew C. Novick, John G. Hissong, Alexandru Almasan and Joseph A. DiDonato
Pages 882 - 888
http://dx.doi.org/10.4161/cbt.3.9.1056
Abstract | PDF

Studies have shown that Resveratrol (RE) can inhibit cancer initiation, promotion, and progression. However the gene expression profile in renal cell carcinoma (RCC) in response to RE treatment has never been reported. To understand the potential anticancer effect of RE on RCC at molecular level, we profiled and analyzed the expression of 2059 cancer-related genes in a RCC cell line RCC54 treated with RE. Biological functions of 633 genes were annotated based on biological process ontology and clustered into functional categories. Twenty-nine highly differentially expressed genes in RE treated RCC54 were identified and the potential implications of some gene expression alterations in RCC carcinogenesis were identified. RE was also shown to inhibit cell growth and induce cell death of RCC cells. The expression alterations of selected genes were validated using reverse transcription polymerase chain reaction. In addition, the gene expression profiles under different RE treatments were analyzed and visualized using singular value decomposition. The findings from this study support the hypothesis that RE induces differential expression of genes that are directly or indirectly related to the inhibition of RCC cell growth and induction of RCC cell death. In addition, it is apparent that the gene expression alterations due to RE treatment depend strongly on RE concentration. This study provides a general understanding of the overall genetic response of RCC54 to RE treatment and yields insights into the understanding of the cancer preventive mechanism of RE in RCC.




COMMENTARY

Open Access Article

Resveratrol: A whiff that induces a biologically specific tsunami

John M. Pezzuto
Pages 889 - 890
http://dx.doi.org/10.4161/cbt.3.9.1129
Abstract | PDF

Commentary to:

Effects of Resveratrol on Gene Expression in Renal Cell Carcinoma

Ting Shi, Louis S. Liou, Provash Sadhukhan, Zhong-Hui Duan, Andrew C. Novick, John G. Hissong, Alexandru Almasan and Joseph A. DiDonato




RESEARCH PAPERS

Open Access Article

Identification of HLA-A2-restricted CTL epitope encoded by the MAGE-n gene of human hepatocellular carcinoma

Hai-Long Dong, Zeng-Shan Li, Jing Ye, Ping Qu, Ya-Yu Huang, Wen Wu, Shao-Ying Lu, Guang-Sheng Chen and Yan-Fang Sui
Pages 891 - 898
http://dx.doi.org/10.4161/cbt.3.9.1081
Abstract | PDF

Background: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1.

Methods: A HLA-A2.1-restricted CTL epitope was identified by using an improved “reverse immunology” strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1.

Results: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro.

Conclusions: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.

Open Access Article

Digital HLA allelotyping

Ozlem Topaloglu, Cur I. Civin and Fred Bunz
Pages 899 - 902
http://dx.doi.org/10.4161/cbt.3.9.1083
Abstract | PDF

Stem cell transplantation is a potentially curative therapy for a number of neoplastic disorders. Successful transplantation of bone marrow, and other sources of stem cells, depends upon the extent to which human leukocyte antigen (HLA) alleles in donor and recipient are matched. HLA incompatibility can result in cytotoxic T-cell mediated graft failure or graft versus host disease. It has been shown that accurate matching between donor and recipient minimizes both of these outcomes and results in improved patient survival. Therefore, methods for high resolution HLA analysis have substantial clinical impact. Heterozygosity at the HLA loci interferes with current methods of DNA sequencebased HLA typing. We present a novel method for HLA analysis, which we term Digital HLA Allelotyping, or dig-HLA. This method employs a strategy in which the expressed HLA alleles are randomly sorted and individually identified.




RESEARCH PHILOSOPHY

Open Access Article

Stagnation and herd mentality in the biomedical sciences

Jonathan R. Brody and Scott E. Kern
Pages 903 - 910
http://dx.doi.org/10.4161/cbt.3.9.1082
Abstract | PDF

Academic biomedical science is like music, painting, or other fashionable arts and politics. Concepts that are perceived to be ‘in’ can become widely accepted and then stagnate, remaining unchallenged for many years, independent of their scientific validity. Fads in biomedical science have been observed to last for years or decades. The reasons for herd mentality and stagnation are manifold, but their recognition allows opportunities for constructive awareness and perhaps effective countermeasures.




PROFILES AND LEGACIES

Open Access Article

Cancer therapeutics: Personal experiences and future perspectives

Enrico Mihich
Pages 911 - 915
http://dx.doi.org/10.4161/cbt.3.9.1144
Abstract | PDF

No abstract available.