The pathogenesis of tuberculosis involves multiple phases and is believed to involve both a carefully deployed series of adaptive bacterial virulence factors and inappropriate host immune responses that lead to tissue damage. A defined Mycobacterium tuberculosis mutant strain lacking the sigH-encoded transcription factor showed a distinctive infection phenotype. In resistant C57BL͞6 mice, the mutant achieved high bacterial counts in lung and spleen that persisted in tissues in a pattern identical to those of wild-type bacteria. Despite a high bacterial burden, the mutant produced a blunted, delayed pulmonary inflammatory response, and recruited fewer CD4 ؉ and CD8 ؉ T cells to the lung in the early stages of infection. In susceptible C3H mice, the mutant again showed diminished immunopathology and was nonlethal at over 170 days after intravenous infection, in contrast to isogenic wild-type bacilli, which killed with a median time to death of 52 days. Complete genomic microarray analysis revealed that M. tuberculosis sigH may mediate the transcription of at least 31 genes directly and that it modulates the expression of about 150 others; the SigH regulon governs thioredoxin recycling and may be involved in the maintenance of intrabacterial reducing capacity. These data show that the M. tuberculosis sigH gene is dispensable for bacterial growth and survival within the host, but is required for the production of immunopathology and lethality. This phenotype demonstrates that beyond an ability to grow and persist within the host, M. tuberculosis has distinct virulence mechanisms that elicit deleterious host responses and progressive pulmonary disease.T uberculosis is one of the leading infectious causes of death and claims Ϸ2 million lives annually (1). There is controversy over whether the disease is primarily a dysfunctional immunologic reaction to a persistent microbe or whether the bacteria themselves produce tissue damage; there is evidence that both host and bacterial factors play key roles in disease severity. In susceptible mouse strains, such as C3H, the pathogen elicits a dysregulated, necrotizing host immune response leading to tissue destruction and further bacterial replication. In resistant mice such as C57BL͞6, Mycobacterium tuberculosis survives in high numbers for many months and is contained in organized granulomatous lesions in the lung without progressive lung damage. Thus whereas mycobacteria survive in both genetic backgrounds, disease progression is delayed in resistant animals (2-4).On the bacterial side, M. tuberculosis virulence has been associated with its initial survival within macrophages and resistance to reactive oxygen and nitrogen intermediates (ROIs and RNIs) (5-8). Tubercle bacilli demonstrate inducible responses to oxidative stresses, and several M. tuberculosis genes, including katG (catalase peroxidase), ahpC (alkylhydroperoxide reductase), and sodA and sodC (superoxide dismutases) have been implicated in protection from the macrophage oxidative burst (9-11). Another potential ...
Little is known about epigenetic silencing of genes by promoter hypermethylation in lobular breast cancers. The promoter methylation status of 5 cancer-related genes (RASSF1A, HIN-1, RAR-, Cyclin D2 and Twist) was evaluated in 2 types of lobular cancers, in situ (LCIS) and invasive lobular carcinomas (ILC) (n ؍ 32), and compared to ductal in situ (DCIS) and invasive (IDC) breast cancers (n ؍ 71). By using methylation-specific PCR (MSP), 100% of ILC and 69% of LCIS cases were found to have 1 or more hypermethylated genes among the panel of 5 genes (compared to 100% IDC and 95% of DCIS). Two or more hypermethylated genes were detected per tumor in 79% of invasive and 61% of in situ lobular carcinomas compared to 81% of IDC and 77% of DCIS. By contrast, DNA from nearly all normal reduction mammoplasty tissues (n ؍ 8) was unmethylated for the 5 genes. The methylation profiles of lobular vs. ductal carcinomas with respect to RASSF1A, Cyclin D2, RAR, and Hin-1 genes were similar, suggesting that gene silencing by promoter hypermethylation is likely to be important in both groups of diseases. Distinctly different, Twist was hypermethylated less often in ILC (16%, 3/19 cases) than in IDC (56%, 15/27 cases) (p ؍ 0.01). These results suggest that these 2 types of tumors share many common methylation patterns and some molecular differences. Additional studies might lend further understanding into the etiology and clinical behavior of this tumor type. © 2003 Wiley-Liss, Inc. Key words: methylation; breast cancer; lobular; ductal; DCIS; LCISComprehensive gene analyses such as serial analysis of gene expression (SAGE) and microarray analysis performed on breast cancer tissues revealed the expression profiles of thousands of genes and resulted in the identification of messenger RNAs that are over-and underexpressed in breast carcinomas compared to normal breast tissue. 1,2 We and others found that a major mode of tumor-specific downregulation of a number of these genes is by DNA hypermethylation. [3][4][5][6][7][8] The hypermethylation of cytosine residues in CpG-rich islands present in the promoter region of genes is an epigenetic alteration that leads to heritable changes in gene expression without changing the DNA sequence, most likely through the formation of repressive chromatin structure. Aberrant methylation of DNA is therefore believed to be an alternative pathway to cancer. 9 Studies of DNA hypermethylation in breast carcinoma have identified certain key genes as targets for epigenetic downregulation, 9 -13 including receptors such as the estrogen receptor 6,14,15 23 Identification of these genes has helped begin to elucidate the molecular pathogenesis of breast carcinoma, as it reveals intracellular pathways that are altered that likely contribute to oncogenesis. In addition, identification of methylated genes provides a potential target for molecular detection of breast carcinoma, as even small amounts of methylated sequences are readily detectable by methylation-specific PCR (MSP). 24 Because of the relat...
If detected early, breast cancer is eminently curable. To detect breast cancer in samples with little cellularity, a high level of sensitivity is needed. Tumor-specific promoter hypermethylation has provided such a valuable tool for detection of cancer cells in biological samples. To accurately assess promoter hypermethylation for many genes simultaneously in small samples, we developed a novel method, quantitative multiplex-methylationspecific PCR (QM-MSP). QM-MSP is highly sensitive (1 in 10 4 -10 5 copies of DNA) and linear over 5 orders of magnitude. For RASSF1A, TWIST, Cyclin D2, and HIN1, we observed significant differences in both the degree (P < 0.003) and incidence (P < 0.02) of hypermethylation between normal and malignant breast tissues. Evaluation of the cumulative hypermethylation of the four genes within each sample revealed a high level of sensitivity (84%) and specificity (89%) of detection of methylation. We demonstrate the application of this technique for detecting hypermethylated RASSF1A, TWIST, Cyclin D2, HIN1, and RARB in 50 -1000 epithelial cells collected from breast ducts during endoscopy or by lavage. Such an approach could be used in a variety of small samples derived from different tissues, with these or different biomarkers to enhance detection of malignancy.
Purpose: Most often it is not the primary tumor, but metastasis to distant organs that results in the death of breast cancer patients. To characterize molecular alterations in breast cancer metastasis, we investigated the frequency of hypermethylation of five genes (Cyclin D2, RAR-, Twist, RASSF1A, and HIN-1) in metastasis to four common sites: lymph node, bone, brain, and lung.Experimental Design: Methylation-specific PCR for the five genes was performed on DNA extracted from archival paraffin-embedded specimens of paired primary breast cancer and its lymph nodes (LN) metastasis (n ؍ 25 each); in independent samples of metastasis to the bone (n ؍ 12), brain (n ؍ 8), and lung (n ؍ 10); and in normal bone, brain, and lung (n ؍ 22).Results: No hypermethylation was detected in the five genes in the normal host tissues. In paired samples, LN metastasis had a trend of higher prevalence of methylation compared with the primary breast carcinoma for all five genes with significance for HIN-1 (P ؍ 0.04). Compared with the primary breast carcinomas, all five genes had higher methylation frequencies in the bone, brain, and lung metastasis, with HIN-1 and RAR- methylation being significantly higher (P < 0.01) in each group. Loss of expression of all five genes correlated, with a few exceptions, to hypermethylation of their promoter sequences in metastatic carcinoma cells microdissected from LNs.Conclusion: The frequent presence of hypermethylated genes in locoregional and distant metastasis could render them particularly susceptible to therapy targeted toward gene reactivation combining demethylating agents, histone deacetylase inhibitors, and/or differentiating agents.
Purpose: To provide a molecular rationale for negative prognostic factors more prevalent in African-American (AA) than Caucasian (Cau) women, we investigated the frequency of promoter hypermethylation in invasive ductal breast cancers in the two races.Experimental Design: HIN-1, Twist, Cyclin D2, RAR-, and RASSF1A were analyzed in DNA from 67 AA and 44 Cau invasive ductal breast cancers, stratified by age and estrogen receptor/progesterone receptor (ER/PR) status, by methylation-specific PCR. Hierarchical multiple logistic regression analysis was applied to determine estimated probabilities of methylation. Expression of HIN-1 mRNA was analyzed by in situ hybridization and quantitative reverse transcribed PCR.Results: Significant differences between races were observed in the ER؊/PR؊, age < 50 subgroup; AA tumors had higher frequency of methylation (P < 0.001) in four of five genes as compared with Cau and also a higher prevalence (80 versus 0%; P < 0.005) of three or more methylated genes per tumor. No differences in gene methylation patterns were observed across the two races for ER؉/PR؉ tumors in all ages and ER؊/PR؊ tumors in age > 50. ER؉/PR؉ status was associated with higher frequency of methylation in Cau tumors of all ages but only with the age > 50 subgroup in AA. Frequent Cyclin D2 methylation was significantly associated (P ؍ 0.01) with shorter survival time.Conclusion: ER؊/PR؊, age < 50 tumors in AA women, have a significantly higher frequency of hypermethylation than in those of Cau women. Comparative studies, such as these, could provide a molecular basis for differences in tumor progression and pathology seen in the two races.
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