Natarajan Ganesan

Disruption of transforming growth factor-β signaling through β-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation

Transforming growth factor-β (TGF-β) signaling members, TGF-β receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf+/− mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-β signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.

γ-Radiation-Induced Chromosomal Mutagen Sensitivity Is Associated with Breast Cancer Risk in African-American Women: Caffeine Modulates the Outcome of Mutagen Sensitivity Assay

Several different cancer studies have indicated that lymphocyte mutagen sensitivity is a marker of DNA repair deficiency and increased cancer risk. We have used a mutagen sensitivity assay (MSA) measuring γ-radiation-induced chromosomal aberrations in freshly cultured lymphocytes and assessed breast cancer risk in African-American women. Concurrently, we conducted duplicate cultures in the presence of caffeine, which overrides G2 arrest in cultured cells, decreases time to DNA repair, and hence increases the aberration rate. In comparison with the non–caffeine-treated cells, we are conceptually segregating the contribution of DNA repair and time for DNA repair as individual susceptibility phenotypes. Blood samples were obtained from 61 cases and 86 controls at Howard University Hospital. Two sets of whole-blood cultures were established and γ-irradiated (1 Gy) at 67 hours, one of which was treated with caffeine (1 mg/mL). Thereafter, cultures were processed for obtaining metaphase spreads. Fifty metaphases were screened for chromatid breaks. The mean breaks per cell (MBPC) for cases (0.34 ± 0.15) was significantly greater than for controls (0.24 ± 0.12; P < 0.0001). Using the 75th percentile value of controls as a cutoff to define mutagen sensitivity, the sensitive individuals had an odds ratio of 4.5 (95% confidence intervals, 2.2-9.1) for breast cancer compared with individuals that were not sensitive. The adjusted odds ratio was 3.3 (95% confidence intervals, 0.147-73.917), which was statistically significant but was limited by the small number of subjects. The results for caffeine co-culture were not predictive of breast cancer (MBPC: cases, 1.6 ± 0.9 versus controls, 1.5 ± 0.8; P = 0.8663). Comparing the MBPC for caffeine and noncaffeine cultures, there was a correlation in controls (n = 79; Spearman r = 0.4286; P < 0.0001), but not in cases (n = 58; Spearman r = 0.06609; P = 0.6221). This study indicates that the MSA phenotype is a risk factor for breast cancer in African-American women, with a significant effect observable even in small studies. The use of caffeine did not enhance the predictivity of MSA, but the correlation with non-caffeine cultures in controls indicates that the MSA phenotype is due to both DNA repair and G2 arrest capacity. (Cancer Epidemiol Biomarkers Prev 2006;15(3):437–42)

Morphological and growth altering effects of Cisplatin in C. albicans using fluorescence microscopy

Changes in morphology and growth curve of Candida albicans in response to treatment by Cisplatin has been studied using fluorescence staining with ethidium bromide. Treatment with Cisplatin was found to markedly inhibit hyphae and ovoid growth as revealed by ethidium bromide staining of drug treated cells. These changes were concomitant with inhibitory effects on the growth curve with respect to untreated cellsPresence of Cisplatin not only caused suppression in the limiting values in the growth curve, but also caused a slight left shift in the EC50 values. Some of the ovoid cells undergoing poisoning with cisplatin were found to be unusually enlarged before undergoing their natural fate thus suggesting formation of similar cytotoxic end products with DNA.

BIOINFORMATICS DATA PROFILING TOOLS: A PRELUDE TO METABOLIC PROFILING

The term metabolic profiling is often used to denote the systematic characterization of the unique biochemical trails or fingerprints left behind by cellular processes. Advances in computational biosciences are often invaluable in dealing with the huge amount of raw data generated from the countless biochemical intermediates that flood the cell at any given time. As a prelude to metabolic profiling, it is essential to completely profile and compile all related information about the genetic and proteomic data. Profiling tools in bioinformatics refer to all those software (web based and downloadable) that compile all related information in single user-interfaces. Generally, these interfaces take a query such as a DNA, RNA, or protein sequence or keyword; and search one or more databases for information related to that sequence. Summaries and aggregate results are provided in a single standardized format that would otherwise have required visits to many smaller sites or direct literature searches to compile. In other words they are software portals or gateways that simplify the process of finding information about a query in the large and growing number of bioinformatics databases.

Abstract 3114: MLL2 overexpression and incomplete proteolytic processing in human cell lines from invasive breast and colon cancers

Background: MLL2 is an epigenetic regulator in mammalian cells and mediates histone 3 lysine 4 tri-methylation (H3K4me3) at specific gene loci, through formation of a multiprotein complex. MLL2 shares a high degree of structural similarity with MLL1, which frequently undergoes translocations resulting in leukemogenic fusion oncoproteins. Despite the structural similarity with MLL1, MLL2 is not involved in leukemias. Earlier we reported aberrant MLL2 expression in human breast and colonic tumor tissues compared to normal tissues. In continuation with the above study, we now present the results of MLL2 investigation in cell lines from human breast and colon cancers. Methods: Established cell lines from human mammary and colonic tissues were cultured under optimal conditions. Cells were processed for nuclear and cytoplasmic fractionation as well as DNA and total RNA extraction. Nuclear and cytoplasmic extracts were separated on polyacrylamide gels and immunoblotted for MLL2. The RNA extracts were used to determine MLL2 transcript levels by one-step reverse transcriptase-PCR using appropriate primers. The DNA extracts were used to amplify MLL2 specific sequences using appropriate primers, followed by sequencing reaction. Result: Examination of cell lines revealed 2-3 fold increase in MLL2 transcript levels in the highly invasive tumor cell lines of both breast and colon. Expression of MLL2 at protein level was consistent with the transcript levels, indicating MLL2 overexpression as the cause of increased protein levels in the tumor cells. Additionally, we also observed incomplete proteolytic processing of MLL2 in the highly invasive cell lines from either tissue types, compared to the corresponding non-tumor/less invasive tumor cell lines. Interestingly, the unprocessed full length MLL2 was significantly abundant in the nuclear compartments of the highly invasive tumor cell lines. Analysis of the coding sequences for the putative cleavage site did not reveal any alterations. Conclusion: We conclude that MLL2 expression and proteolytic processing are both deregulated in breast and colon cancers. This deregulation may be associated with mammary and colonic tumor growth/progression. Given the epigenetic regulatory role, further investigation is required to study MLL2 involvement in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3114.