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Human hepatocarcinogenesis is a complex process with many unresolved issues, including the cell of origin (differentiated and/or progenitor/stem cells) and the initial steps leading to tumor development. With the aim of providing new tools for studying hepatocellular carcinoma initiation and progression, we developed an innovative model based on primary human hepatocytes (PHHs) lentivirus-transduced with SV40LT+ST, HRASV12 with or without hTERT. The differentiation status of these transduced-PHHs was characterized by RNA sequencing (including lncRNAs), and the expression of some differentiation markers confirmed by RT-qPCR and immunofluorescence. In addition, their transformation capacity was assessed by colony formation in soft agar and tumorigenicity evaluated in immune-deficient mice. The co-expression of SV40LT+ST and HRASV12 in PHHs, in association or not with hTERT, led to the emergence of transformed clones. These clones exhibited a poorly differentiated cell phenotype with expression of stemness and mesenchymal-epithelial transition markers and gave rise to cancer stem cell subpopulations. In vivo, they resulted in poorly differentiated hepatocellular carcinomas with a reactivation of endogenous hTERT. These experiments demonstrate for the first time that non-cycling human mature hepatocytes can be permissive to in vitro transformation. This cellular tool provides the first comprehensive in vitro model for identifying genetic/epigenetic changes driving human hepatocarcinogenesis.

International audience; BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most frequent human cancers and a major cause of cancer-related death worldwide. The major risk factors for developing HCC are infection by hepatitis B virus (HBV) and hepatitis C virus (HCV), chronic alcoholism, and aflatoxins; however, critical gene targets remain largely unknown. Herein, we sought to establish DNA methylation patterns in HCC and corresponding cirrhotic tissues and to identify DNA methylation changes associated with major risk factors. METHODS: We have established assays for quantitative analysis of DNA methylation levels in a panel of seven cancer-associated genes and repetitive elements, and combined these assays with a series of HCC tumors, associated with major risk factors, collected from two different geographical areas. RESULTS: We found a high frequency of aberrant hypermethylation of specific genes (RASSF1A, GSTP1, CHRNA3, and DOK1) in HCC tumors as compared to control cirrhotic or normal liver tissues, suggesting that aberrant hypermethylation exhibits non-random and tumor-specific patterns in HCC. Importantly, our analysis revealed an association between alcohol intake and the hypomethylation of MGMT and between hypermethylation of GSTP1 and HBV infection, indicating that hypermethylation of the genes analyzed in HCC tumors exhibits remarkably distinct patterns depending on associated risk factors. CONCLUSIONS: This study identifies aberrant DNA methylation of specific cellular genes in HCC and the major risk factors associated with these changes, providing information that could be exploited for biomarker discovery in clinics and molecular epidemiology.

Cervical cancer that is associated with high-risk human papillomaviruses (HPV) is the most common malignancy in Indian women. Therefore, the establishment of a prevention program is urgently required considering both vaccination and screening. However, relatively little is known about the prevalence of the different HPV types in cervical cancers in different regions of India, particularly central India. In this study, we have determined the HPV type distribution in 180 cervical cancers of women from Sevagram, a rural area from central India. In addition, we have analyzed other epithelial cancers that are known to be in part associated with high-risk HPV infection, i.e. oral (n=65), vulva and vagina (n=7) and penis (n=7). Approximately 93% of cervical cancers were positive for one or more of the high-risk HPV types. HPV16 was the predominant type being present in 81.7% of the cases. The remaining most predominant high-risk HPV types were: 18, 31, 35, 45, 56 and 59. Oral and vulva/vagina cancers were exclusively associated with HPV16, 27.7% and 70%, respectively. None of penile cancers was found positive for any of the high-risk HPV types. These data show that HPV16 vaccination in this geographical region will have considerable impact on the prevention of cervical and other epithelial cancers.

Epidemiological studies have demonstrated that 15 different mucosal human papillomavirus (HPV) types of the genus alpha of the HPV phylogetic tree are classified as high risk for cervical cancer development. Three additional HPV types of the same genus, HPV26, 53 and 66, are classified as probable high-risk types. In this study, we have characterized the biological properties of the E7 oncoproteins from these three HPV types. All of the corresponding E7 proteins were able to associate with retinoblastoma protein (pRb) and up-regulated the expression of several positive cell cycle regulators, i.e. CDK2, cyclin A and cylin E. However, HPV26 E7 appears to be more efficient than HPV53 and 66 E7 in up-regulating the transcription of cyclin A. Unlike E7 from the high-risk type HPV16 protein, HPV26, 53 and 66 did not efficiently promote pRb degradation. In addition, E7 from these viruses was able to promote proliferation of primary human keratinocytes and circumvent G1 arrest imposed by overexpression of p16 INK4a , but with less efficiency than the high-risk HPV16 E7. Together, our data show that in vitro properties of these E7 proteins correlate with the epidemiological classification of HPV26, 53 and 66 as HPV types with an intermediate risk for cervical cancer development.

Human Papillomavirus type 77 is a skin type found in non-melanoma skin cancers of immuno-compromised individuals. Although, the HPV77 E6 oncoprotein has been well studied, nothing is known about E7. Studies on mucosal HPV types (e.g. HPV16) showed that E7 deregulates the cell cycle by binding to and promoting degradation of retinoblastoma protein (pRb). Here, we characterized the impact of HPV77 E7 on the cell cycle. We observed that HPV77 E7 associated with pRb with a lower affinity than HPV16 E7, promoting weakly its degradation. Although, HPV16 E7 led to cellular proliferation and accumulation of the cell cycle inhibitor p16 INK4a , both events were not clearly observed in HPV77 E7 cells. Together, these data indicate that HPV77 E7 does not efficiently deregulate the cell cycle, in contrast to several E7s of mucosal HPV types.

Chronic inflammation induced by biological, chemical and physical factors has been associated with increased risk of human cancer at various sites. Inflammation facilitates the initiation of normal cells and their growth and progression to malignancy through production of pro-inflammatory cytokines and diverse reactive oxygen and nitrogen species. These also activate signaling molecules involved in inflammation and carcinogenesis such as nuclear transcription factor (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Several chemopreventive agents act through inhibition of signaling pathways (e.g. NF-κB), inhibition of oxidant-generating enzymes (e.g. iNOS) and mediators of inflammation (e.g. COX-2), scavenging reactive oxygen and nitrogen species, and modulation of xenobiotic-metabolizing enzymes (especially phase II enzyme induction). Some anti-inflammatory drugs have been tested in clinical trials to prevent human cancer at several sites. Better understanding of the molecular mechanisms by which chronic inflammation increases cancer risk will lead to further development of new strategies for cancer prevention at many sites.

The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNE1 ) was localized to the E3–F3 region of mouse chromosome 2 and to the q11–q15 region of human chromosome 15. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNE1 in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 15. Clinically, the human SGNE1 DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 15q11–15q13 region.

Cyp cells are permissive murine cells carrying a thermosensitive polyoma virus genome that remains integrated at 39 degrees C, but is effectively excised and replicated after transfer to 33 degrees C. In rare subclones of the Cyp line, temperature shift-down yields predominantly homogeneous populations of chimeric molecules that appear to reflect the circularization of defined segments of DNA spanning one of the junctions between the integrated viral genome and the adjacent cellular DNA. Such accurate and frequent excision requires a specific recombination mechanism. We examined both the cellular and the viral sequences that cross-over to generate one of these chimeric molecules, Rm I. The homology at the cross-over site is one of 1 or 2 base pairs at most; patches of homology, amounting in total to 19 or 20 base pairs, are found in perfect register on both sides of this site; and the two stretches of DNA that are joined to form RM I contain similar 12-14 base pair sequences (5'- CTCCTTTACAGAGG -3' and 5'- CTCCTTTCAAGG -3') in opposite orientations.

Excision (and replication) of the integrated viral DNA is induced when mouse cells transformed by an early thermosensitive mutant of polyoma (Py) virus (Cyp cells) are transferred from the restrictive temperature of 39° at which they are propagated, to that of 33°. In a number of clones derived from the Cyp line, the low-molecular-weight DNA generated after such an induction is represented almost exclusively by nondefective, monomeric, Py genomes (5.3 kb). In one clone, however, this DNA was found to consist predominantly of copies of a cyclic molecule of approximately 7.1 kb (Rm I). This molecule was shown to include sequences colinear with most, if not the whole, of Py DNA, as well as other sequences that presumably originated from those flanking the integrated viral DNA. The data reported here demonstrate that Rm I is a hybrid molecule comprising 1.03 copies of Py DNA and about 1.6 kb of mouse DNA, joined in a unique fashion: whereas the mouse DNA insertion terminates with an imperfect inverted repeat of 7 bp, it is connected with viral by 3273 at one end, and with viral by 3092 at the other end. As Rm I is colinear with Py DNA from by 3092 to 5292/1, and from by 1 to by 3273, it follows that the cellular insertion is flanked by a direct viral repeat of 182 bp. These results strongly suggest that a selective mechanism, necessarily distinct from nonspecific homologous recombination, is involved in the excision of Py DNA in induced Cyp cells.