e-Lynx - May 2008

Dear Researcher,

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Technical Report

Chromatin, Epigenetics and Cancer
by Judd C. Rice - Article Provided by Active Motif.

Cancer, which arises when “normal” cells in the body begin to divide out of control, is the second leading cause of deaths in the United States. It is currently estimated that half of all men and a third of all women in the U.S. will develop cancer during their lifetimes. While the “War on Cancer” was declared nearly four decades ago, only recently have new basic and clinical scientific insights led to innovative approaches into how many cancers can be treated and cured. These studies were founded on the common underlying theme that the DNA in each cell is packaged and organized into a structure known as chromatin. Chromatin ultimately affects any DNA-templated process ranging from gene expression to cell division and, importantly, the molecular mechanisms that regulate chromatin are often disrupted early during cancer progression. Therefore, further study of chromatin biology is essential to provide novel targets and development of future therapeutic interventions for human diseases, including cancer.

Chromatin and Histone
The three billion base pairs of DNA in each of your cells are incorporated into chromatin by associating with a group of small evolutionarily conserved proteins, the histones. Two molecules each of the core histone proteins (H2A, H2B, H3 and H4) form an octamer where ~147 bp of DNA wraps around it to create the fundamental repeating subunit of chromatin, the nucleosome. A specialized histone (linker histone H1) functions to further compact the tens of millions of nucleosomes into more highly ordered structures. It is this compacted DNA-protein complex that serves as the template for all DNA-dependent processes including DNA replication, repair, recombination, chromosome segregation and gene expression. It has long been known that the histones are subject to a variety of post-translational modifications (phosphorylation, acetylation, ubiquitylation, methylation: two different types – arginine and lysine) and that these modifications play important roles in chromatin structure and function (see page 12 for a summary). With regard to transcriptional regulation, increases in histone acetylation and specific methylation events (H3 lysine 4 & lysine 36 methylation) are generally associated with increased gene expression, while decreased acetylation and other histone methylation events (H3 lysine 9 and lysine 27 methylation) are hallmarks of decreased gene expression (See ref. 6 for a recent review)

Epigenetics and DNA Methylation
Epigenetics refers to the study of heritable changes in phenotypes that have no underlying changes in genotype. In other words, daughter cells take on altered characteristics of the mother cell without changing their DNA sequence, usually by changing their overall gene expression patterns. The best studied epigenetic phenomenon is the methylation of cytosine within cytosine-guanine dinucleotides (CpGs), also known as DNA methylation. CpG dinucleotides are relatively scarce in the genome but tend to cluster in “islands” which are usually located in the 5’ regulatory regions of about half of all human genes. These islands are typically unmethylated, however, during cancer progression, many become aberrantly methylated; an event intimately associated with the repression of critical tumor suppressor genes. The patterns of DNA methylation are maintained during cell division giving rise to daughter cells with a vastly enhanced potential to become cancer cells.

“How do chromatin biology and
epigenetics intersect in the
development of cancer, and
what can be done to
treat cancers involving
deleterious epigenetic events?”

Initially epigenetics was thought only to be associated with DNA methylation however an updated definition takes into account other models of gene regulation and incorporates recent advances in chromatin biology, including histone modifications (2). Changes in gene expression patterns associated with altered states of DNA methylation and histone modification can persist through many cell divisions, so any such change in expression meets the current definition of an epigenetic event. So, the question arises: how do chromatin biology and epigenetics intersect in the development of cancer, and what can be done to treat cancers involving deleterious epigenetic events?

Role of Chromatin Modifications in Cancer Etiology
Chromatin-based mechanisms exist within the cell to partition specific regions of the genome into those that are transcriptionally active and those that are repressed. This is a necessary event in order to prevent inappropriate gene expression patterns and to maintain cellular identity. When an inappropriate change in DNA methylation and/or chromatin modifications leads to aberrant gene regulation, this is referred to as an ‘epimutation’ or epigenetic mutation. The epigenetic nature of cancer arises from the fact that, frequently, these chromatin-based mechanisms are high-jacked in cancer cells to facilitate unchecked cell growth by silencing cell cycle control genes and activating genes that promote growth. But whether these epigenetic events are causal to the development of cancer or contribute to a secondary effect (or both) remains uncertain.
It is becoming increasingly clear that global and local changes in histone modifications and DNA methylation events are not necessarily mutually exclusive. While it was previously believed that epimutation began with DNA methylation, a recent revised paradigm strongly suggests that alterations in histone modifications occur early to influence gene expression (4). At this point the modifications, especially changes in histone acetylation, and changes in gene expression patterns are reversible. Other modifications include the demethylation of lysine 4 of histone H3 and the methylation of lysine 9 (and possibly lysine 27). It is only when these promoters acquire methylated DNA does the expression of the gene become “locked” down and the combined epimutation becomes stable and heritable.

Epigenetic Therapy as an Emerging Treatment Model for Cancer
The overall goal of epigenetic therapy is to re-activate the silenced tumor suppressor genes leading to a cessation of cell division and cancer growth. Currently, the two major types of epigenetic intervention aim to 1) reverse DNA methylation by inhibiting the DNA methyltransferase (DNMT) enzyme(s), and 2) to increase histone acetylation by inhibiting the histone deacetylase (HDAC) enzymes that remove this modification. A number of compounds that inhibit these enzymes have already been identified and, in pre-clinical trials, shown to effectively re-activate gene expression and halt cancer growth. Dozens of HDACs and DNMT inhibitors are currently part of a number of clinical trials aimed to develop regimens best suited for treating specific cancers (see table 1). Two drugs, Zolinza (aka vorinostat, SAHA, developed by Merck) and Vidaza (5-azacytidine, developed by Pharmion) have already been approved by the FDA for use in treating specific types of cancer (Zolinza for cutaneous T-cell lymphoma and Vidaza for myelodysplastic syndrome). Despite the success of these drugs when used independently, it is likely that the key to unlocking the real potential of these drugs to successfully treat cancer lies within combination therapy with each other or with other established chemotherapeutic agents. The order of addition of HDAC or DNMT inhibitors in combination with standard chemotherapies may determine the response of the cancer to the therapy, whether the cancer cells exit the cell cycle, undergo apoptosis or halt division and differentiate. For example, HDAC inhibitors sensitize cancer cells to subsequent chemotherapy, stimulating them to undergo programmed cell death.

Due to their initial success at halting cancer growth, many companies and institutions have initiated high throughput drug discovery programs dedicated to identifying new epigenetic therapeutics. Since methylation of lysines 9 and possibly 27 of histone H3 are involved in the epigenetic silencing of genes, the enzymes that catalyze these reactions (Suv39h1/2, EZH2) are also prime targets for inhibition in these programs.

The Use of Epigenetics in the Detection, Prognosis and Treatment of Cancer
While treatment of cancer as an epigenetic disease and using agents that target the machinery of chromatin modification, it is also possible that epigenetics may lead to the prevention or early detection of cancer. Promoter CpG hypermethylation at a number of genes has been correlated with the occurrence or predisposition to cancer (e.g. hypermethylation of p16 gene in the lung epithelia of heavy smokers; see ref. 7 for review). DNA methylation has a number of advantages for use as a biomarker. It is particularly straightforward to detect, the modification is very stable, and DNA methylation is amenable to automated high-throughput analysis. Histone modifications lag in these regards, primarily because the normal states for histone modifications remain to be fully mapped in normal tissues to provide a baseline of comparison to identify changes that correlate with the development of cancer. Some interesting correlations have been made, though.

• EZH2 is overexpressed in metastatic prostate cancer (9, 10)
• Reduction in levels of trimethyl H4 lysine 20 and trimethyl H3 lysine 9 trimethyl are observed in many malignancies (1, 3)
• Prostate cancer recurrence risk correlated with IHC staining for histone modifications (5, 8)

Future Directions
There has been good progress toward understanding the mechanisms of epigenetics and the role they play in cancer, but further research is obviously critical to the continued development of effective treatments of the disease. There are certainly more issues to be addressed than can be mentioned here, but these are some very pressing questions that must be answered to further the understanding of the epigenetic basis of cancer:

• What initiates the epigenetic lesion at a tumor
suppressor gene?
• Why are hematologic cancers more responsive to HDAC and DNMT inhibitors than solid tissue cancers?
• Do HDAC inhibitors have off-target (non-histone) effects that are relevant to the therapeutic outcome?
• What assays best serve as readouts for the efficacy of epigenetic therapies?
• What other classes of chromatin modifying proteins can be targeted for therapeutic intervention?

TABLE: Some of the major epigenetic therapies currently in clinical trials: HDAC inhibitors (shaded purple), DNA methylation inhibitors (shaded rose) or trials involving both (shaded green). Other names of the compounds listed are in parentheses. Abbreviations- AML: acute myelogenous leukemia; ATRA: all-trans retinoic acid; BCC: basal cell carcinoma; CLL: chronic lymphocytic leukemia; CML: chronic myelogenous leukemia; CTCL: Cutaneous T-cell Lymphoma; CRC: Colorectal cancer; FAP: Familial adenomatous polyposis; HRPC: Hormone-refractory prostate cancer; MDS: myelodysplastic syndrome; MM: Multiple myeloma; NHL: non-Hodgkin’s Lymphoma; NSCLC: Non small-cell lung cancer; PC: prostate cancer; PTCL: peripheral T-cell lymphoma; RCC: renal cell carcinoma; VPA: valproic acid