The identification of recurrent somatic mutations in genes encoding epigenetic enzymes has provided a strong rationale for the development of compounds that target the epigenome for the treatment of cancer. This notion is supported by biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases to promoter regions through association with oncogenic fusion proteins such as PML-RARα and AML1-ETO. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis; however, it remains unclear why tumor cells are more sensitive to HDACi-induced cell death than normal cells. Herein, we assessed the biological and molecular responses of isogenic normal and transformed cells to the FDA-approved HDACi vorinostat and romidepsin. Both HDACi selectively killed cells of diverse tissue origin that had been transformed through the serial introduction of different oncogenes. Time-course microarray expression profiling revealed that normal and transformed cells transcriptionally responded to vorinostat treatment. Over 4200 genes responded differently to vorinostat in normal and transformed cells and gene ontology and pathway analyses identified a tumor-cell-selective pro-apoptotic gene-expression signature that consisted of BCL2 family genes. In particular, HDACi induced tumor-cell-selective upregulation of the pro-apoptotic gene BMF and downregulation of the pro-survival gene BCL2A1 encoding BFL-1. Maintenance of BFL-1 levels in transformed cells through forced expression conferred vorinostat resistance, indicating that specific and selective engagement of the intrinsic apoptotic pathway underlies the tumor-cell-selective apoptotic activities of these agents. The ability of HDACi to affect the growth and survival of tumor cells whilst leaving normal cells relatively unharmed is fundamental to their successful clinical application. This study provides new insight into the transcriptional effects of HDACi in human donor-matched normal and transformed cells, and implicates specific molecules and pathways in the tumor-selective cytotoxic activity of these compounds.

Human papilloma viruses (HPVs) are accepted as being carcinogenic in human cervical and anogenital cancers. The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV types 16 and 18. For this investigation, DNA that had been previously extracted and fresh frozen at À701C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. We show that HPV 18 gene sequences are present in DNA extracted from breast tumours in Australian women. Overall, 24 (48%) of the 50 samples were HPV positive. Overall no correlations with tumour grade, patient survival, steroid receptor status, ERB-2, p53 expression and mutation were observed. Human papilloma viruses may have a role in human breast cancer. We speculate that HPVs may be transmitted by hand from the female perineum to the breast. It is accepted that human papillomavirus (HPV) types 16 and 18 are carcinogenic, and that probably HPV types 31 and 33 are also carcinogenic in human cervical and anogenital cancers (IARC, 1995). The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV 16 and 18 (Band et al, 1990;De Villiers et al, 2005).Human papilloma virus 16 has been identified in breast tumours in Italian women and breast tumours in Norwegian women who had previous cervical neoplasia (Hennig et al, 1999). Human papilloma virus 33 has been identified in breast cancer in Chinese and Japanese women (Yu et al, 1999) MATERIALS AND METHODSFor this investigation, DNA that had been previously extracted and fresh frozen at À70 O C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. The DNA samples were amplified twice per sample using GenomiPhit DNA Amplification Kit (Amersham Biosciences). The DNA quality was confirmed by PCR, amplifying 268 bp of the b-globin gene. These samples were then screened for the presence of HPV by PCR using primers that could detect 140 bp in the E6 region of HPV 16, 18 and 33 (Yu et al, 1999). DNA extracted from cervical cancer cell lines HeLa and SiHa cells were used as positive controls for HPV 18 and 16, respectively. Plasmid plink 322 HPV 33 was used as a positive control for HPV 33. DNA from leukaemia Raji cells was used a negative control. The PCR products were separated on 7.6% PAGE and visualised by SYBR Green I (Molecular Probes). The screening was repeated five times using different batches of DNA samples amplified by the GenomiPhit DNA Amplification Kit (Amersham Biosciences). Human papilloma virus-positive samples were sequenced (there was sufficient material to sequence 18 of 24 HPV-positive samples).Grade of tumour and survival of patients were known for each sample. Screening for exons 5...

Sir,High-risk human papilloma viruses (HPVs) have been consistently identified in 13 -86% of breast tumours in the 10 studies published since 1999 (Lawson et al, 2006). High-risk HPVs of the same type have been identified in both cervical and breast cancer that had occurred in the same women (Hennig et al, 1999;Widschwendter et al, 2004). This observation has lead to the hypothesis that HPVs may be transmitted to the breast during sexual activities (Kan et al, 2005). If this hypothesis is correct, it is likely that HPV-positive breast cancers would occur in women younger than those with HPV-negative breast cancer. This is because HPV genital infections are much more common in young women who have had multiple sexual partners (IARC, 1995).There are only two studies in which the age of women with HPV-positive and -negative breast cancer has been published. There were no differences in the average of age of women with either HPV-positive and -negative breast cancer in a study of Brazilian women (Damin et al, 2004). This is in contrast to a recent study of Greek women in which those with HPV-positive breast cancer were of average age 38 years as compared to average age 53 years for women with HPV-negative breast cancer (P-values for difference ¼ 0.001) (Kroupis et al, 2006).We have reviewed the ages of Australian women with HPVpositive and -negative breast cancer in our study published in this Journal (Kan et al, 2005). These data are shown in Table 1. The average age of women with HPV-positive breast cancer was 55.6 years as compared to 63.8 years for women with HPV-negative breast cancer (P-values for difference ¼ 0.049). These data are compatible with the hypothesis that HPV-positive breast cancers occur in younger women than those with HPV-negative breast cancers, and that high-risk HPVs may have been transmitted by sexual activity with HPV-positive sexual partners. HPV ¼ human papilloma virus. P-value for difference in average ages ¼ 0.049, which is significant at the 95% level. REFERENCES

The immortalization process is a fundamental step in the development of most (if not all) human cancers, including the aggressive endothelial cell (EC)-derived malignancy angiosarcoma. Inactivation of the tumor suppressor p16INK4a and the development of multiple chromosomal abnormalities are features of angiosarcoma that are recapitulated during telomerase-mediated immortalization of human ECs in vitro. The present study used a panel of telomerase-immortalized bone marrow EC (BMEC) lines to define the consequences of inactivation of p16INK4a on EC function and to identify molecular changes associated with repression of p16INK4a. In a comparison of two immortalized BMEC mass cultures and six clones, the cell lines that repressed p16INK4a showed a higher rate of proliferation and an impaired ability to undergo morphogenic differentiation and form vessel-like structures in vitro. Proteomic comparison of a p16INK4a-negative and a p16INK4a-positive BMEC mass culture at early- and late-passage time points following transduction with telomerase reverse transcriptase (hTERT) revealed altered expression of cytoskeletal proteins, including vimentin and α-tropomyosin (αTm), in the immortal cells. Immunoblot analyses of a panel of 11 immortal clones showed that cells that lacked p16INK4a expression tended to accumulate more dramatic changes in these cytoskeletal proteins than cells that retained p16INK4a expression. This corresponded with aberrant cytoskeletal architectures among p16INK4a-negative clones, which featured thicker actin stress fibers and less fluid membrane ruffles than p16INK4a-positive cells. A direct link between p16INK4a repression and defective EC function was confirmed by analysis of normal cells transfected with small interfering RNA (siRNA) targeting p16INK4a. siRNA-mediated repression of p16INK4a significantly impaired random motility and vessel formation in vitro. This report is the first to demonstrate that ECs that repress the expression of p16INK4a are prone to defects in motility, morphogenesis and cytoskeletal organization. These defects are likely to reflect alterations that occur during the development of EC-derived malignancies.