Little is known about the alterations in microRNA (miRNA) expression patterns during the consecutive stages of cervical cancer development and their association with chromosomal instability. In this study, miRNA expression in normal cervical squamous epithelium, high-grade precancerous lesions (cervical intraepithelial neoplasia (CIN2-3)), squamous cell carcinomas (SCCs) and adenocarcinomas (AdCAs) was integrated with previously generated chromosomal profiles of the same samples. Significantly differential expression during the consecutive stages of cervical SCC development was observed for 106 miRNAs. Of these differentially expressed miRNAs, 27 showed early transiently altered expression in CIN2-3 lesions only, 46 miRNAs showed late altered expression in SCCs only and 33 showed continuously altered expression in both CIN2-3 and SCCs. Altered expression of five significantly differentially expressed miRNAs, hsa-miR-9 (1q23.2), hsa-miR-15b (3q25.32), hsa-miR-28-5p (3q27.3), hsa-miR-100 and hsa-miR-125b (both 11q24.1), was directly linked to frequent chromosomal alterations. Functional analyses were performed for hsa-miR-9, representing a potential oncogene with increased expression linked to a chromosomal gain of 1q. Hsa-miR-9 overexpression was found to increase cell viability, anchorage-independent growth and migration in vitro. Upon organic raft culturing, hsa-miR-9 hampered differentiation and induced proliferation in all strata of the epithelial layer. These findings support a potential oncogenic function of hsa-miR-9 in cervical cancer. In summary, differential expression of 106 miRNAs, partly associated with chromosomal alterations, was observed during cervical SCC development. Altered expression of hsa-miR-9 associated with a chromosomal gain of chromosome 1q was shown to be functionally relevant, underlining the importance of deregulated miRNA expression in cervical carcinogenesis.

No abstract

In lymph node (LN) proliferation centers in chronic lymphocytic leukemia (CLL), the environment protects from apoptotic and cytotoxic triggers. Here, we aimed to define the molecular basis for the in-

Chronic lymphocytic leukemia (CLL) cells circulating in peripheral blood (PB) differ from the leukemic fraction in lymph nodes (LNs) with respect to cell division and drug sensitivity. CD40 stimulation of PB CLL cells in vitro results in chemoresistance and provides a partial model for the LN microenvironment. The TLR9 ligand CpG induces proliferation in immunoglobulin variable heavychain-unmutated CLL, but apoptosis in immunoglobulin variable heavy-chain-mutated CLL. To juxtapose proliferative with antiapoptotic signals, we investigated the effects of CpG in the context of CD40 ligation in mutated versus unmutated CLL cells in this study. Prolonged CD40 ligation induced classical, followed by alternative nuclear factor-jB (NF-jB), activity in both subgroups, correlating with enhanced Bfl-1 and Bcl-X L levels, respectively. A dichotomy in NF-jB signaling occurred on combined CD40/TLR9 triggering. This induced declining p52 and Bcl-X L levels, and reversed chemoresistance only in mutated cells, whereas unmutated cells proliferated, maintained p52 and Bcl-X L and remained chemoresistant. The pivotal contribution of Bcl-X L to chemoresistance was shown by the BH3 mimetic ABT-737 and RNA interference. Finally, in ex vivo LN samples, p52, p65 and Bcl-X L levels were highly expressed, corroborating the in vitro findings. Thus, a distinction in NF-jB activation and drug susceptibility in mutated versus unmutated (LN-like) CLL cells was uncovered, which was causally linked to Bcl-X L levels.

Recently, it has been described that patients with chronic lymphocytic leukemia (CLL) have increased numbers of regulatory T (T(reg)) cells. In the present study, we analysed the mechanism behind T(reg) cells expansion in CLL. Neither analysis of the T-cell receptor repertoire nor CD45 isoform expression of T(reg) cells from patients with CLL provided evidence for chronic (tumor) antigenic stimulation as a possible cause for T(reg) cells expansion in CLL. We found evidence however for increased formation of T(reg) cells via CD70 costimulation, because we observed that CD40 ligand activated CLL cells (which might be considered a model of lymph node CLL cells) strongly induced CD70-dependent formation of T(reg) cells. Reverse transcription-multiplex ligation-dependent probe amplification assay expression analysis of 34 apoptosis-regulating genes showed that in comparison with other CD4(+) T-cells, T(reg) cells from both healthy individuals (HD) and patients with CLL had a high expression of pro-apoptotic Noxa and a low expression of anti-apoptotic Bcl-2. Strikingly, Bcl-2 levels of T(reg) cells in patients with CLL were significantly higher than in HD. Finally, the different apoptotic profile resulted in differences at the functional level, because T(reg) cells from patients with CLL were more resistant to drug-induced apoptosis than T(reg) cells from HD. In conclusion, T(reg) cells in CLL may accumulate both by increased formation, facilitated by CD27-CD70 interaction in the lymph node proliferation centres, and decreased sensitivity to apoptosis because of a shifted Noxa-Bcl-2 balance.

SummaryTo enhance the poor antigen-presenting capacity of B-cell chronic lymphocytic leukaemia (B-CLL), CD40 triggering has been considered as an active immunotherapy. However, CD40 stimulation also has an antiapoptotic effect and may further impair the dysregulated response of B-CLL to apoptotic stimuli. Therefore, we measured the expression of virtually all regulators of apoptosis before and after CD40 stimulation. These findings were correlated with sensitivity for chemotherapy-and death-receptorinduced apoptosis and T-cell-mediated killing. CD40 stimulation enhanced the constitutive anti-apoptotic profile of B-CLL cells by upregulation of Bcl-xL and Bfl-1 and downregulation of the BH3-only protein Harakiri. Unexpectedly, the BH3-only protein Bid was strongly induced. Functionally, CD40-stimulated B-CLL cells became resistant to drug-induced apoptosis and, despite upregulation of CD95 and Bid, were not sensitive to CD95L. In contrast, autologous T cell killing, triggered by loading CLL cells with viral (CMV) peptides, was very efficient both before and after CD40 stimulation. Upon CTL interaction, CLL targets underwent mitochondrial depolarization and caspase-3 activation. Thus, despite an increased anti-apoptotic profile, CD40 triggered B-CLL cells remain excellent targets for resident cytotoxic T cells. These data support therapeutic exploitation of CD40 stimulation in B-CLL, provided that a strong CTL component is induced.

Deregulated expression of microRNAs (miRNAs) is common and biologically relevant in cervical carcinogenesis and appears only partly related to chromosomal changes. We recently identified 32 miRNAs showing decreased expression in high-grade cervical intraepithelial neoplasia (CIN) and carcinomas not associated with a chromosomal loss, 6 of which were located within a CpG island. This study aimed to investigate to what extent these miRNAs are subject to DNA methylation-mediated transcriptional repression in cervical carcinogenesis.   Methylation-specific PCR (MSP) analysis on a cell line panel representing different stages of human papillomavirus (HPV) induced transformation revealed an increase in methylation of hsa-miR-149, -203 and -375 with progression to malignancy, whereas expression of these miRNAs was restored upon treatment with a demethylating agent. All three miRNAs showed significantly increased levels of methylation in cervical carcinomas, whereas methylation levels of hsa-miR-203 and -375 were also significantly increased in high-grade CIN. A pilot analysis showed that increased hsa-miR-203 methylation was also detectable in HPV-positive cervical scrapes of women with high-grade CIN compared with controls. Similar to recent findings on hsa-miR-375, ectopic expression of hsa-miR-203 in cervical cancer cells decreased both the proliferation rate and anchorage independent growth. We found evidence for methylation-mediated transcriptional repression of hsa-miR-149, -203 and -375 in cervical cancer. Methylation of the latter two was already apparent in precancerous lesions and represent functionally relevant events in HPV-mediated transformation. Increased hsa-miR-203 methylation was detectable in scrapes of women with high-grade CIN, indicating that methylated miRNAs may provide putative markers to assess the presence of (pre)cancerous lesions.

Although accumulating evidence indicates that chronic lymphocytic leukemia (CLL) is a disease with appreciable cell dynamics, it remains uncertain whether this also applies to patients with stable disease. In this study, 2 H 2 O was administered to a clinically homogeneous cohort of nine stable, untreated CLL patients. CLL dynamics in blood and bone marrow were determined and compared with normal B-cell dynamics in blood from five healthy individuals who underwent a similar 2 H 2 O labeling protocol. Average CLL turnover rates (0.08-0.35% of the clone per day) were f2-fold lower than average B-cell turnover rates from healthy individuals (0.34-0.89%), whereas the rate at which labeled CLL cells in blood disappeared (0.00-0.39% of B cells per day) was f10-fold lower compared with labeled B cells from healthy individuals (1.57-4.24% per day). Leukemic cell turnover variables inversely correlated with the level of somatic hypermutation of the CLL clone (IgVH mutations). Although CLL cells in bone marrow had a higher level of label enrichment than CLL cells in blood, no difference between proliferation rates and proapoptotic and antiapoptotic profiles of CLL cells from these compartments was observed. These data suggest that, in stable disease, there is a biological relationship between the degree of somatic hypermutation of the CLL clone and its dynamics in vivo. Furthermore, in contrast to lymph nodes, the bone marrow does not seem to be a major CLL proliferation site. [Cancer Res 2008;68(24):10137-44]