Assessment of the kinetics of Treponema pallidum dissemination into blood and tissues in experimental syphilis by real-time quantitative PCR.

Little is known about the size and kinetics of treponemal burdens in blood and tissues during acquired or experimental syphilitic infection. We used real-time quantitative PCR to measure Treponema pallidum DNA levels in rabbits infected intratesticularly with the prototype Nichols strain. At the outset, we performed a series of in vitro blood spiking experiments to determine the effect of blood processing procedures on the distribution of treponemes in various blood components. T. pallidum DNA levels in plasma and whole blood were approximately 10-fold higher than those in serum and more than 200-fold greater than those in peripheral blood mononuclear cells (PBMCs). Ten rabbits were inoculated intratesticularly with doses of treponemes ranging from 4 x 10(7) to 2 x 10(8) organisms. In five rabbits, T. pallidum DNA levels were measured sequentially in serum, plasma, whole blood, and PBMCs until sacrifice at peak orchitis, at which time brain, kidney, liver, spleen, and testicles were harvested; blood and organs were also harvested at orchitis from the other five rabbits. T. pallidum DNA was detected in plasma within 24 h postinfection. Treponeme levels in whole blood and blood components increased significantly with the development of peak orchitis. Overall, levels in serum and PBMCs were lower than those in plasma and whole blood; this disparity was particularly marked at early time points. Significantly greater numbers of spirochetes were found in the spleen than in liver, kidney, or brain tissue at the time of sacrifice. Our findings highlight the remarkable capacity of T. pallidum to disseminate from the site of infection to blood and tissues, and they identify the spleen as a prime target for treponemal invasion.

Aberrant methylation of the cyclin D2 promoter in primary small cell, nonsmall cell lung and breast cancers.

DNA methylation alteration of several genes contributes to human tumorigenesis. Cyclin D2, a member of the D-type cyclins, is implicated in cell cycle regulation and malignant transformation. In our study, we examined the methylation status of the cyclin D2 promoter in small cell lung cancer (SCLC), nonsmall cell lung cancer (NSCLC), breast tumors and tumor cell lines. We observed that aberrant methylation of cyclin D2 was present in 32 of 56 (57%) SCLC cell lines, 7 of 32 (22%) SCLC tumor tissues; 25 of 61 (47%) NSCLC cell lines, 19 of 48 (40%) NSCLC tumor tissues; 18 of 30 (60%) breast tumor cell lines and 19 of 63 (30%) breast tumor tissues. Methylation was more frequent in the tumor cell lines compared to the primary breast and SCLC tumors (p = 0.007 and p = 0.001, respectively). Methylation was rare in the control tissue samples; 0 of 12 peripheral blood lymphocytes; 0 of 12 buccal epithelial cells; 0 of 18 nonmalignant lung tissues and 3 of 28 (11%) nonmalignant breast tissues. Promoter methylation correlated with loss of transcript by reverse transcription PCR (RT-PCR) in 9 of 11 (6 lung, 5 breast) tumor cell lines tested. Two cell lines that were not methylated also lacked expression, suggesting that other mechanisms of inactivation may be involved. Expression was restored by treatment with the demethylating agent, 5 aza 2' deoxycytidine, in all 9 methylated cell lines. Our results confirm earlier reports in breast cancer and indicate that aberrant methylation of cyclin D2 may contribute to the pathogenesis of the 2 major types of lung cancers.

Aberrant methylation of the HIC1 promoter is a frequent event in specific pediatric neoplasms.

PURPOSE: To determine the role of methylation of HIC1, a candidate tumor suppressor gene on 17p13.3, in various types of pediatric tumors. EXPERIMENTAL DESIGN: We examined the methylation status of the HIC1 promoter by methylation specific PCR in 157 pediatric tumors and 27 nonmalignant tissues. We correlated methylation with mRNA expression by reverse transcription-PCR in eight tumor-derived cell lines. RESULTS: HIC1 methylation was frequent in medulloblastomas (80%, 12 of 15), retinoblastomas (67%, 6 of 9), rhabdomyosarcomas (59%, 13 of 22), germ cell tumors (55%, 6 of 11), and neurouroblastic tumors (36%, 14 of 39); neuroblastomas (43%, 12 of 28); ganglioneuromas (17%, 1 of 6); and ganglioneuroblastomas (20%, 1 of 5). In contrast, a low incidence of methylation was observed in osteosarcomas (17%, 2 of 12), Ewing's tumors (9%, 1 of 11), Wilms' tumors (3%, 1 of 31), and hepatoblastomas (0%, 0 of 7). HIC1 methylation was more frequent in the aggressive alveolar subtype of rhabdomyosarcomas (100%, 8 of 8) than the embryonal subtype (33%, 4 of 12; P < 0.005) and was rare in the nonmalignant tissues examined. Methylation was also demonstrated by sequencing in nine randomly selected tumor samples. Seven of eight pediatric tumor cell lines examined were methylated and showed loss or reduced HIC1 mRNA. Expression was strongly induced in all cell lines by treatment with the demethylating agent 5-aza 2'deoxycytidine. CONCLUSIONS: Our data suggest that aberrant methylation of HIC1 may play a role in the pathogenesis of specific pediatric tumors.

Aberrant methylation of TMS1 in small cell, non small cell lung cancer and breast cancer.

TMS1 (target of methylation-induced silencing) is a CpG island-associated gene that functions in the regulation of apoptosis and encodes a caspase recruitment domain, a recently described motif found in apoptotic signaling molecules. Recent evidence suggests that silencing of genes in the apoptotic pathway contribute to human carcinogenesis. We examined the DNA methylation status of the TMS1 promoter in lung and breast tumor tissues, tumor cell lines and nonmalignant tissues by methylation-specific polymerase chain reaction (MSP) and its mRNA expression by reverse transcription PCR. Aberrant methylation of TMS1 was present in 70% (40 of 57) of small cell lung cancer (SCLC) cell lines and 41% (13 of 32) of SCLC tumor tissues, 48% (29 of 61) of non small cell lung cancer (NSCLC) cell lines and 40% (28 of 70) of NSCLC tumor tissues and 46% (12 of 26) of breast cancer cell lines and 32% (20 of 63) of breast tumor tissues. Methylation was absent in the peripheral blood lymphocytes and buccal epithelium from healthy volunteers, as well as in nonmalignant lung tissues and was rare in nonmalignant breast tissues 7% (2 of 30). DNA methylation was confirmed by sequence analysis and the methylation status correlated inversely with TMS1 RNA expression in 18 cell lines tested. RNA expression was restored by treatment with the demethylating agent 5-aza-2'-deoxycytidine, in 4 of 4 methylated cell lines that lacked the TMS1 transcript. Our results suggest that methylation of TMS1 may play a role in the pathogenesis of small cell and non small lung and breast cancers.

Methylation profiles of sporadic ovarian tumors and nonmalignant ovaries from high-risk women.

PURPOSE: The purpose of this research was to examine the DNA methylation profiles of primary sporadic ovarian cancers and ovarian tissues from high-risk women. EXPERIMENTAL DESIGN: We analyzed the DNA methylation status of nine cancer-related genes in 49 primary ovarian tumors, 39 nonmalignant ovarian tissues obtained from 16 women with no known risk and from 23 high-risk women with a strong family history of breast and/or ovarian cancer or BRCA1 germ-line mutations, and 11 ovarian cancer cell lines, by methylation-specific PCR. RESULTS: Our findings are as follows: (a) methylation rates of four of nine genes, RASSF1A (41%), HIC1 (35%), E-cadherin (29%), and APC (18%) were significantly higher in tumors compared with controls. At least one of the four genes was methylated in 76% of the tumors; (b) a low frequency of methylation was present in nonmalignant tissues; (c) no significant differences in methylation frequencies were seen between the nonmalignant ovarian tissues from women at high-risk and those with no known risk of developing ovarian cancer; (d) methylation of the BRCA1 gene was found in 10% of sporadic tumors but in none of the samples from women with a germ-line BRCA1 mutation; and (e) ovarian cancer cell lines showed a similar frequency of methylation to ovarian tumors except for the HIC1 gene. CONCLUSIONS: Our results suggest that aberrant methylation of specific genes, including two not described previously, may be important in ovarian cancer pathogenesis but not in ovaries at risk for cancer development.