Richard B. Roth

Gene expression analyses reveal molecular relationships among 20 regions of the human CNS

Transcriptional profiling was performed to survey the global expression patterns of 20 anatomically distinct sites of the human central nervous system (CNS). Forty-five non-CNS tissues were also profiled to allow for comparative analyses. Using principal component analysis and hierarchical clustering, we were able to show that the expression patterns of the 20 CNS sites profiled were significantly different from all non-CNS tissues and were also similar to one another, indicating an underlying common expression signature. By focusing our analyses on the 20 sites of the CNS, we were able to show that these 20 sites could be segregated into discrete groups with underlying similarities in anatomical structure and, in many cases, functional activity. These findings suggest that gene expression data can help define CNS function at the molecular level. We have identified subsets of genes with the following patterns of expression: (1) across the CNS, suggesting homeostatic/housekeeping function; (2) in subsets of functionally related sites of the CNS identified by our unsupervised learning analyses; and (3) in single sites within the CNS, indicating their participation in distinct site-specific functions. By performing network analyses on these gene sets, we identified many pathways that are upregulated in particular sites of the CNS, some of which were previously described in the literature, validating both our dataset and approach. In summary, we have generated a database of gene expression that can be used to gain valuable insight into the molecular characterization of functions carried out by different sites of the human CNS.

The adaptive imbalance in base excision-repair enzymes generates microsatellite instability in chronic inflammation

Chronic infection and associated inflammation are key contributors to human carcinogenesis. Ulcerative colitis (UC) is an oxyradical overload disease and is characterized by free radical stress and colon cancer proneness. Here we examined tissues from noncancerous colons of ulcerative colitis patients to determine (a) the activity of two base excision-repair enzymes, AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease; and (b) the prevalence of microsatellite instability (MSI). AAG and APE1 were significantly increased in UC colon epithelium undergoing elevated inflammation and MSI was positively correlated with their imbalanced enzymatic activities. These latter results were supported by mechanistic studies using yeast and human cell models in which overexpression of AAG and/or APE1 was associated with frameshift mutations and MSI. Our results are consistent with the hypothesis that the adaptive and imbalanced increase in AAG and APE1 is a novel mechanism contributing to MSI in patients with UC and may extend to chronic inflammatory or other diseases with MSI of unknown etiology.

Human endometriosis is associated with plasma cells and overexpression of B lymphocyte stimulator

Endometriosis affects 10–20% of women of reproductive age and is associated with pelvic pain and infertility, and its pathogenesis is not well understood. We used genomewide transcriptional profiling to characterize endometriosis and found that it exhibits a gene expression signature consistent with an underlying autoimmune mechanism. Endometriosis lesions are characterized by the presence of abundant plasma cells, many of which produce IgM, and macrophages that produce BLyS/BAFF/TNFSF13B, a member of the TNF superfamily implicated in other autoimmune diseases. B lymphocyte stimulator (BLyS) protein was found elevated in the serum of endometriosis patients. These observations suggest a model for the pathology of endometriosis where BLyS-responsive plasma cells interact with retrograde menstrual tissues to give rise to endometriosis lesions.

Large-Scale Association Study Identifies ICAM Gene Region as Breast and Prostate Cancer Susceptibility Locus

We conducted a large-scale association study to identify genes that influence nonfamilial breast cancer risk using a collection of German cases and matched controls and >25,000 single nucleotide polymorphisms located within 16,000 genes. One of the candidate loci identified was located on chromosome 19p13.2 [odds ratio (OR) = 1.5, P = 0.001]. The effect was substantially stronger in the subset of cases with reported family history of breast cancer (OR = 3.4, P = 0.001). The finding was subsequently replicated in two independent collections (combined OR = 1.4, P < 0.001) and was also associated with predisposition to prostate cancer in an independent sample set of prostate cancer cases and matched controls (OR = 1.4, P = 0.002). High-density single nucleotide polymorphism mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes ICAM1, ICAM4, and ICAM5. Although genetic variants in ICAM5 showed the strongest association with disease status, ICAM1 is expressed at highest levels in normal and tumor breast tissue. A variant in ICAM5 was also associated with disease progression and prognosis. Because ICAMs are suitable targets for antibodies and small molecules, these findings may not only provide diagnostic and prognostic markers but also new therapeutic opportunities in breast and prostate cancer.

Suppression of RAD21 gene expression decreases cell growth and enhances cytotoxicity of etoposide and bleomycin in human breast cancer cells

A genome-wide case-control association study done in our laboratory has identified a single nucleotide polymorphism located in RAD21 as being significantly associated with breast cancer susceptibility. RAD21 is believed to function in sister chromatid alignment as part of the cohesin complex and also in double-strand break (DSB) repair. Following our initial finding, expression studies revealed a 1.25- to 2.5-fold increased expression of this gene in several human breast cancer cell lines as compared with normal breast tissue. To determine whether suppression of RAD21 expression influences cellular proliferation, RNA interference technology was used in breast cancer cell lines MCF-7 and T-47D. Proliferation of cells treated with RAD21-specific small inhibitory RNA (siRNA) was significantly reduced as compared with mock-transfected cells and cells transfected with a control siRNA (Lamin A/C). This inhibition of proliferation correlated with a significant reduction in the expression of RAD21 mRNA and with an increased level of apoptosis. Moreover, MCF-7 cell sensitivity to two DNA-damaging chemotherapeutic agents, etoposide and bleomycin, was increased after inhibition of RAD21 expression with a dose reduction factor 50 (DRF50) of 1.42 and 3.71, respectively. At the highest concentrations of etoposide and bleomycin administered, cells transfected with a single siRNA duplex targeted against RAD21 showed 57% and 60% survival as compared with control cells, respectively. Based on these findings, we conclude that RAD21 is a novel target for developing cancer therapeutics that can potentially enhance the antitumor activity of chemotherapeutic agents acting via induction of DNA damage.

Gene transfer to suppress bone marrow alkylation sensitivity.

Alkylating agents represent a highly cytotoxic class of chemotherapeutic compounds that are extremely effective anti-tumor agents. Unfortunately, alkylating agents damage both malignant and non-malignant tissues. Bone marrow is especially sensitive to damage by alkylating agent chemotherapy, and is a dose-limiting tissue when treating cancer patients. One strategy to overcome bone marrow sensitivity to alkylating agent exposure involves gene transfer of the DNA repair protein O(6)-methylguanine DNA methyltransferase (O(6)MeG DNA MTase) into bone marrow cells. O(6)MeG DNA MTase is of particular interest because it functions to protect against the mutagenic, clastogenic and cytotoxic effects of many chemotherapeutic alkylating agents. By increasing the O(6)MeG DNA MTase repair capacity of bone marrow cells, it is hoped that this tissue will become alkylation resistant, thereby increasing the therapeutic window for the selective destruction of malignant tissue. In this review, the field of O(6)MeG DNA MTase gene transfer into bone marrow cells will be summarized with an emphasis placed on strategies used for suppressing the deleterious side effects of chemotherapeutic alkylating agent treatment.

Technology challenges in screening single gene disorders.

The completion of the human genome project and the accelerated discovery of genes responsible for single gene disorders will allow for the preventive screening of carriers and patients. Accuracy and reliability of analytic tests are major demands on technologies used in a diagnostic screening environment. The consistency of results and the potential of multiplexing suggest mass spectrometry as the method of choice for large-scale genetic screening programs. The added ability to analyze other large molecules such as peptides and proteins further underlines the versatility and usefulness of this technology. We describe the use of mass spectrometry for screening of sickle cell disease at the level of DNA and protein. Conclusion:analytic methods are needed for large-scale screening and diagnostic applications that are highly accurate, fully automated and cost-effective. The precise measurement of molecular weights and the use of high fidelity enzymes to produce diagnostic products make mass spectrometry the method of choice for DNA and protein screening procedures.