Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines

We describe novel effects of p53 loss on immortal transformation, based upon comparison of immortally transformed human mammary epithelial cell (HMEC) lines lacking functional p53 with closely related p53(+) lines. Our previous studies of p53(+) immortal HMEC lines indicated that overcoming the stringent replicative senescence step associated with critically short telomeres (agonescence), produced indefinite lifespan lines that maintained growth without immediately expressing telomerase activity. These telomerase(-) 'conditionally immortal' HMEC underwent an additional step, termed conversion, to become fully immortal telomerase(+) lines with uniform good growth. The very gradual conversion process was associated with slow heterogeneous growth and high expression of the cyclin-dependent kinase inhibitor p57(Kip2). We now show that p53 suppresses telomerase activity and is necessary for the p57 expression in early passage p53(+) conditionally immortal HMEC lines, and that p53(-/-) lines exhibit telomerase reactivation and attain full immortality much more rapidly. A p53-inhibiting genetic suppressor element introduced into early passages of a conditionally immortal telomerase(-) p53(+) HMEC line led to rapid induction of hTERT mRNA, expression of telomerase activity, loss of p57 expression, and quick attainment of uniform good growth. These studies indicate that derangements in p53 function may impact malignant progression through direct effects on the conversion process, a potentially rate-limiting step in HMEC acquisition of uniform unlimited growth potential. These studies also provide evidence that the function of p53 in suppression of telomerase activity is separable from its cell cycle checkpoint function.     

Immortalization of oral keratinocytes by functional inactivation of the p53 and pRb pathways

A subgroup of head and neck squamous cell carcinomas (HNSCCs) contains high‐risk human papillomavirus‐type 16 (HPV16). The viral E6 and E7 oncoproteins inactivate the p53 and pRb proteins, respectively. We examined the causative effect of HPV16 E6 and E7 expression on the immortalization of normal oral keratinocytes (OKCs) and compared the resulting phenotype with alternative ways of p53‐ and pRb‐pathway abrogation frequently found in HNSCCs without HPV. Primary OKCs were conditionally immortalized with temperature‐sensitive SV40 large T‐antigen and human telomerase, allowing these cells to return to their senescent primary state after temperature shift. HPV16 E6 and E7 were introduced to overcome senescence, determined with population doubling (PD) as read‐out. For comparison, we downregulated p53 and p16 by short hairpin RNA genes and expressed mutant p53R(175)H and cyclinD1. Expression of HPV16 E6 caused an extended life span similar to expression of mutant p53R(175)H or p53 knockdown. Expression of mutant p53R(175)H seemed to cause additional activation of the hypoxia and WNT signaling pathways. HPV16 E7 expression had no direct effect on lifespan, similar to p16 knockdown or cyclinD1 expression. In combination with HPV16 E6 or other functional inactivations of p53, abrogation of the pRb‐pathway by either HPV16 E7 or other manipulations caused an immortal phenotype. Our data show the causative role of HPV16 E6/E7 in early squamous carcinogenesis. Activity of each gene could be mimicked by other genetic events frequently found in HNSCC without HPV. This data provides the experimental proof of causal association of HPV in HNSCC carcinogenesis and further support the crucial role of the p53‐ and pRb‐pathways.     

Spontaneous immortalization of clinically normal colon-derived fibroblasts from a familial adenomatous polyposis patient

Normal human diploid cells do not spontaneously immortalize in culture, but instead enter replicative senescence after a finite number of population doublings. Ablation of key checkpoint arrest or cancer-suppressor genes, through dominantly inherited germline mutation (p53+/-, Li-Fraumeni) or viral oncogene expression (SV40 large T, HPV16/18, and E6/E7) can lead to escape from senescence, additional doublings, and entrance into crisis phase, where immortal clones emerge at low frequency. In the vast majority of cases, telomerase is reactivated and telomeres are stabilized. Here we describe the spontaneous immortalization of clinically normal fibroblasts derived from colonic stroma of a familial adenomatous polyposis (FAP) patient. The preimmortal (C26C) and the spontaneously immortalized derivative (C26Ci) cells are heterozygous for a characterized germline mutation in exon 15 of the adenomatous polyposis coli gene. Immortalization was accompanied by spontaneous reactivation of endogenous telomerase and establishment of telomeres at presenescent lengths. Normal checkpoint behavior is retained and a diploid karyotype is maintained. These cells provide a valuable new addition to the limited number of spontaneously immortalized human cell types, particularly fibroblast cells, and will be useful in experimentally determining the functional pathways in neoplastic development and in the identification of potential molecular targets for cancer chemoprevention.     

Functional interaction of p53 with HPV18 E6, c-myc and H-ras in 3T3 cells.

Wild-type (wt) p53 has been suggested to be the product of a tumor-suppressor gene. Recently, it has been shown that the E6 oncoproteins of human papillomavirus (HPV) types 16 and 18, like the SV40 large T antigen, are physically associated with wt p53. We have investigated the functional interaction of wt p53 with the viral oncogene products of HPV16 and 18 and with cellular oncogenes by transfection of NIH3T3 cells with p53 wt alone or with several oncogene(s). We found that over-expression of HPV18 E6, c-myc or activated H-ras, like SV40 large T, can partially overcome the growth-inhibitory effect of wt p53 in NIH3T3 cells, while HPV16 E6 and E7, HPV18 E7, k-fgf, c-fos and mutant (mt) p53 do not. Further studies indicate that HPV18 E6 and c-myc can overcome the antiproliferative effect, but not the antitransforming effect, of wt p53, while activated H-ras can overcome both the antiproliferative and antitransforming effects of wt p53. These data show evidence of a functional interaction between HPV18 E6 and wt p53, and suggest that the cooperation of HPV E6 and cellular oncogenes c-myc and H-ras, which are activated in several cases of human cervical cancers, may be necessary to overcome completely the anti-oncogenic function of p53 in the development of these tumors.     

A human vascular endothelial cell model to study angiogenesis and tumorigenesis

Endothelial cell biology has recently been the subject of considerable interest in thrombosis and cancer research. However, the successful establishment of immortalized human endothelial cells which retain differentiated cell characteristics has been rare. We have successfully established immortalized human umbilical vein endothelial cells (HUVECs) by human papilloma virus (HPV)-16 E6-E7. HPV-16 E6, E7 and E6- E7 were successfully introduced into HUVEC cells. Both E6 and E7 cultures had an extended lifespan but eventually underwent senescence. E6-E7 cultures 4-5-2G, however, acquired an indefinite lifespan in culture but did not undergo malignant conversion. Telomerase activity was not detected in either E6 or E7 cultures; however, telomerase was detected in E6-E7 4-5-2G cells. The cells exhibited a 'cobblestone' morphology and developed a capillary-like tube structure upon reaching confluence. The 4-5-2G line expressed Factor VIII related antigen and took up DiI-Ac-LDL as markers of endothelial origin. The line expressed integrin subunits (alpha(v)beta3, alph(v)beta5, beta1, alpha2, alpha3, beta4 and alpha6) consistent with an endothelial origin. The higher passage of 4-5-2G line showed a similar intensity of integrin immunostaining to that of primary HUVECS. Subsequent infection of these immortal cells with the Kirsten murine sarcoma virus which contains an activated K-ras oncogene induced morphological transformation that led to the acquisition of invasion capability and neoplastic properties. Telomerase was also detected in the tumorigenic v-Ki-ras transformed cell line. These cell lines should be useful for studies of the molecular mechanisms underlying normal and neoplastic endothelial cell proliferation and migration, and might also provide an in vitro model for development of pharmacologic and gene therapy for cardiovascular thrombosis and cancer.      

Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion

Using an estrogen-inducible retroviral system, we demonstrate that oncogenic Raf-1 induces growth arrest and morphological changes in finite lifespan human mammary epithelial cells (HMEC). This arrest does not rely on expression of the cyclin-dependent kinase inhibitor (CKI) p16(INK4a), nor on changes in expression of the CKIs p21(Cip1), p14(ARF), p27(Kip1) or p57(Kip2). The Raf-induced arrest is independent of viral oncogene mediated inactivation of p53 and pRB, or c-myc overexpression. Flow cytometric analysis demonstrates that cells arrest in both G1 and G2. The Raf-induced arrest is mitigated or eliminated in some immortally transformed HMEC. Immortal HMEC that have both overcome replicative senescence and undergone the recently described conversion process maintain growth in the presence of transduced oncogenic Raf-1; they also gain EGF-independent growth and a low frequency of anchorage-independent growth. However, HMEC that have overcome replicative senescence but have not undergone conversion and HMEC immortalized by transduction with the catalytic subunit of telomerase, hTERT, remain severely growth arrested. These results indicate that the molecular mechanisms responsible for the Raf-1-induced growth arrest may vary among different finite lifespan cell types, and that in HMEC, this mechanism is altered during the conversion process, rather than as a direct consequence of overcoming senescence or expressing hTERT.     

端粒酶在人胚肾上皮细胞转化中的作用

目的:利用原代人胚肾上皮细胞转化模型阐明端粒酶在细胞癌变过程中的作用。方法:用逆转录病毒介导的基因导入法,使人端粒酶催化亚基(humantelomerasecatalyticsubunit,hTERT)、HRas癌基因、猿猴病毒40(simianvirus40,SV40)编码的早期抗原在原代人胚肾上皮细胞中稳定地表达,观察细胞的生长特性、染色体畸变率以及细胞转化特征。结果:端粒酶的激活虽然能延长细胞的寿命,但不能使细胞永生化。如果细胞同时表达端粒酶和SV40编码的大T抗原(largeTantigen,LT),细胞就能获得永生化。在此永生化细胞株中导入HRas癌基因以及SV40编码的小T抗原(smallTantigen,ST),细胞发生恶性转化,表现为在软琼脂上形成克隆并在裸鼠皮下形成肿瘤。与原代细胞相比,永生化细胞株的染色体畸变率无改变,而恶性转化细胞的畸变率明显增加。此外在转化细胞和几种肿瘤细胞中表达阻抑人端粒酶的特异性siRNA,能显著地抑制肿瘤细胞的生长、诱导细胞凋亡并减少软琼脂上形成的克隆数目。结论:细胞永生化是癌变过程的必要阶段,端粒酶的激活不仅是细胞永生化的重要环节,而且在维持肿瘤细胞生长中起重要的作用。     

Production of spindle cell carcinoma by transduction of H-Ras 61L into immortalized human mammary epithelial cells

Human mammary epithelial cells (HMEC) were immortalized by serial passaging through senescence (M0) and subsequent transduction with the catalytic subunit of the human telomerase gene (hTERT). These cells acquired multiple non-random cytogenetic abnormalities with lengthy passaging in vitro, but are still not tumorigenic in irradiated nude mice and cannot grow in soft agar. Transduction, of late passage immortal HMEC from a single donor, with a retroviral vector containing the mutant autoactive H-Ras 61L gene, enabled immortal HMEC to acquire anchorage independent growth properties. Three colonies were picked and all three were found to be tumorigenic. One colony exclusively produced epithelial tumors in nude mice, but the other two colonies gave rise exclusively to malignancies in which the cells displayed a spindle morphology. In this paper we describe the characteristics of the tumors arising from one of these 'spindle colonies'. These tumors were strongly positive for vimentin staining and virtually negative for pan-cytokeratin staining, on immunohistochemistry. Cytogenetic analysis of the cells derived from these tumors confirmed that they were derived from the original cultured, immortalized mammary cells. We conclude that the HMEC have undergone metaplastic transformation due to the high levels of H-Ras 61L and telomerase activity that they display, and the derived tumors are best described as spindle cell carcinomas.     

Association of decreased intercellular communication with the immortal but not the tumorigenic phenotype in human mammary epithelial cells

Intercellular communication was compared in early passage cultures of human mammary epithelial cells (HMEC) from normal and malignant breast tissues and immortalized nontumorigenic human breast cell lines (184A1 and 184B5). A clonogenic assay for the cell-mediated transfer of toxic metabolites of 6-thioguanine (TG) between cells was used as a measure of intercellular communication. We examined the effects of wild-type TG-sensitive (TGs) HMEC density on the survival of mutant TG-resistant (TGr) immortalized HMEC in TG-containing medium. Survival rates of TGr HMEC cocultured with TGs normal HMEC, malignant HMEC, or immortalized nontumorigenic HMEC were dependent on the density of TGs cells. For example, the percentage of recovery of TGr cells cocultured with 10(5) TGs immortalized, normal, or carcinoma HMEC was 88 +/- 4, 41 +/- 10, or 2.0 +/- 1.7, respectively. Gap junction-mediated intercellular communication between homologous HMEC types was also studied, as quantitated on the basis of Lucifer yellow dye transfer between cells in culture. Results from the dye transfer studies supported those from the metabolic cooperation studies. These results using nonimmortalized tumor cells differ from previous reports in which immortal tumor cells have been found to communicate less than their normal counterparts. Previous reports suggesting that tumor cell lines communicate less than normal cells may have resulted from the confounding influence of the immortal phenotype on the tumor phenotype.     

SV40 infection induces telomerase activity in human mesothelial cells

Mesotheliomas are malignant tumors of the pleural and peritoneal membranes which are often associated with asbestos exposure and with Simian virus 40 (SV40) infection. Telomerase activity is repressed in somatic cells and tissues but is activated in immortal and malignant cells. We evaluated telomerase activity in seven primary malignant mesothelioma biopsies and matched lung specimens and 20 mesothelioma cell lines and eight corresponding primary tumor cultures. All the tumor biopsies, and nearly all primary cell mesothelioma cultures and cell lines were telomerase positive. The findings in cell lines paralleled those observed in primary cultures in cases where paired samples were available. Next, we found that SV40, a DNA tumor virus present in approximately 50% of mesothelioma biopsies in the USA, induced telomerase activity in primary human mesothelial cells, but not in primary fibroblasts. Telomerase activity became detectable as early as 72 h following wild-type (strain 776) SV40 infection, and a clear DNA ladder was detectable 1 week after infection. The amount of telomerase activity increased during passage in cell culture and appeared to parallel increases in the cellular amounts of the SV40 large T-antigen. Thus, SV40 infection leads to telomerase activity before the infected mesothelial cells become transformed and immortalized. SV40 infection of human fibroblasts did not cause detectable telomerase activity. We also determined that the SV40 small t-antigen (tag) plays an important role in inducing telomerase activity because this activity was undetectable or minimal in mesothelial cells infected and/or transformed by SV40 tag mutants. Asbestos alone did not induce telomerase activity, and asbestos did not influence telomerase activity in mesothelial cells infected with SV40. Induction of telomerase activity by SV40 may be related to the very high rate of mesothelial cell immortalization that is characteristically associated with SV40 infection of mesothelial cells.     

Functional similarity between HPV16E7, SV40 large T and adenovirus E1a proteins

We have analysed the immortalizing function of Human Papillomavirus type 16 (HPV16) in a rat cell line which has been derived by immortalizing rat embryo fibroblasts with a thermolabile SV40 large T antigen and is temperature sensitive for growth. Introduction of wild type SV40 large T antigen or the adenovirus E1a 12s gene product has previously been shown to readily overcome the inability of these cells to divide at the non-permissive temperature. In contrast, the introduction of myc, another known immortalizing gene, cannot complement the growth defect of these cells. This cell line therefore provides a novel assay system which can distinguish two groups of immortalizing oncogenes. We have shown here that expression of HPV16 can readily complement the growth defect in this rat cell line and that this function can be genetically localised to E7. Cells expressing HPV16 E6 sequences are also weakly rescued from growth arrest at the non-permissive temperature. These results demonstrate a functional similarity between HPV16 E7, SV40 large T and adenovirus E1a and suggest that these genes may immortalize cells by a common mechanism. Interestingly, the limited sequence homology between these three gene products is restricted to the domain which has recently been implicated in binding the retinoblastoma gene product.     

HPV E6癌基因诱导宫颈癌发生机制研究进展

HPV E6和E7癌基因与宫颈癌密切相关,其中E6癌基因在宫颈癌的发生、发展过程中起重要作用.HPV-16 E6蛋白能诱导端粒酶的表达,增加角质形成细胞的端粒酶活性,提示端粒酶的活性在细胞永生化和恶变过程中起重要作用,端粒酶的激活是细胞转化的起始步骤.HPV E6癌基因增加细胞端粒酶活性的机制是激活了端粒酶催化亚基hTERT的转录活性.但HPV癌基因突变导致染色体改变、杂合子丢失、原癌基因的激活在宫颈致癌作用中同样起重要作用.针对E6癌基因诱导宫颈癌发生的分子生物学机制,综述近年来E6癌基因与宫颈癌关系方面的研究进展.     

Mammary epithelial cell transformation: insights from cell culture and mouse models

Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely – a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16^INK4a, p21, p27 and p57), p14^ARF, telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere models has led to the identification of putative stem cells, but more studies are needed to define their biologic role and potential as precursor cells for distinct breast cancers. The combined use of transformation strategies in cell culture and mouse models together with molecular definition of human breast cancer subtypes should help to elucidate the nature of breast cancer diversity and to develop individualized therapies.     

Stimulation of epithelial membrane antigen expression by transforming growth factor-beta in normal and oncogene-transformed human mammary epithelial cells

Transforming growth factor-beta (TGF beta) appears to play a role in regulating the expression of tissue-specific proteins in human mammary epithelial cells (HMEC), regardless of the state of malignant transformation. We demonstrated this by utilizing a series of normal, immortalized, and oncogene-transformed (SV40 T and v-Ha-ras) HMEC derived from one individual, and assaying for expression of a milk fat globule/epithelial membrane antigen (EMA) reactive with E29/EP1 monoclonal antibody. EMA was increased by TGF beta in all HMEC examined. This effect appeared to be dose-dependent between 5 and 15 ng/ml in the normal, immortalized, and v-Ha-ras-transformed cells but saturated at 5 ng/ml in the SV40 T-transformed cells. The SV40 T-transformed cells showed both enhanced basal expression of EMA and increased sensitivity to TGF beta stimulation of EMA expression. The degree of increase in EMA induced by TGF beta was proportional to the level of basal expression in each cell type and appeared to be unrelated to either the number of high affinity TGF beta receptors, or the relative sensitivity to growth inhibition by TGF beta. Therefore, the TGF beta effect on EMA appears to be modulated at a level beyond receptors in these cells. EMA expression was also stimulated by sodium butyrate and dexamethasone (both differentiating agents) in some of the HMEC. The effect of butyrate on EMA expression is consistent with previous findings in which butyrate selectively enhanced production of milk fat globule antigens in the breast tumor cell line MCF-7, ZR-75-1, MDA-MB-134, and MDA-MB-468 cells. The coupled effects of TGF beta and SV40 T-transformation in enhancing the expression of EMA, and the possible effect of SV40 T increasing the responsiveness to TGF beta, may provide a new model for the study of the effect of growth factors in regulating specific gene expression in human breast epithelial cells.     

HPV16-E6上调MIF对宫颈癌细胞增殖和凋亡的影响

目的 检测人乳头瘤病毒16型的致癌基因E6（HPV16-E6）通过巨噬细胞移动抑制因子（MIF）对宫颈癌细胞增殖和凋亡的影响。方法 用HPV16-E6过表达和干扰质粒转染能分泌MIF的人宫颈癌细胞C33A、SiHa和Caski。Western blot和荧光定量PCR法检测MIF蛋白和mRNA表达,并用ELISA法检测培养上清液中MIF蛋白量;将转染后的宫颈癌细胞与人巨噬细胞非接触性共培养,检测巨噬细胞中MIF表达;C33A转染HPV16-E6后加或不加MIF抑制剂,CCK-8法和流式细胞术检测细胞增殖、周期和凋亡情况;采用Pearson相关分析法分析HPV16-E6和MIF间的关系、Kaplan-Meier分析HPV16-E6对患者生存率的影响。结果 癌细胞、上清液和巨噬细胞中MIF表达随HPV16-E6表达而上调（P<0.05）;HPV16-E6过表达可诱导C33A细胞增殖、减少G0/G1期细胞和抑制细胞凋亡;抑制MIF后,细胞增殖率下降、 凋亡率增加（P<0.05）;HPV16-E6表达与MIF正相关（P<0.05）,Kaplan-Meier分析显示HPV16-E6表达与患者总生存率和无进展生存率有关（P<0.05）。结论 HPV16-E6可诱导宫颈癌细胞及其微环境中巨噬细胞MIF表达,并可通过MIF诱导宫颈癌细胞增殖和抑制细胞凋亡而影响宫颈癌的预后。     

The cell biology of human papillomavirus transformed cells

Viruses are becoming increasingly recognized as a major etiological agent in the development of numerous forms of human cancer. Human papillomaviruses (HPVs) have been associated with a number of neoplastic lesions, most notably cervical cancer which is one of the major forms of cancer world wide. Of the over 50 types of identified HPVs, HPV types 16, 18, 31 and 33 are the types most commonly associated with malignant carcinomas. These viruses contain double stranded DNA which code for about eight gene products, some of which are oncogenic when introduces into cultured rodent or human cells. In particular, both the E6 and E7 gene products have different oncogenic capabilities and these genes are selectively retained within the genome of cervical carcinoma derived cells. The E7 gene product has immortalizing capabilities in primary cells and is able to cooperate with an activated ras oncogene to fully transform primary rodent cells. The E7 gene product from HPV type 16 is also capable of complexing in vitro to the anti-oncogene product, Rb. Similar complexes occur with Adenovirus E1A and SV40 large T proteins which may suggest a shared mechanism of transformation used by HPV type 16, Adenovirus and SV40. Transformation studies using primary human cells and nontumorigenic HeLa/fibroblast hybrid cells have also suggested that chromosome 11 may be important in suppressing the HPV transformed phenotype. The transformed phenotype may therefore also involve an impaired intracellular control of persisting HPV oncogenic sequences. Although there exists no solid evidence that a cytotoxic T-lymphocyte reaction is mounted against HPV transformed cells, there is evidence that both NK cells and activated macrophages can preferentially kill HPV transformed cells in vitro. Future studies are required to identify possible targets present on HPV transformed cells which are absent on normal cells.     

Knockdown of p53 combined with expression of the catalytic subunit of telomerase is sufficient to immortalize primary human ovarian surface epithelial cells

Ovarian cancer is developed from a single layer of thin epithelial cells covering the surface of ovary, named human ovarian surface epithelial cells. Like all primary human cells, human ovarian surface epithelial cells have a finite life span and will go into senescence and eventually die when cultured in vitro. Immortalized human ovarian surface epithelial cells will provide an important model system with which to study ovarian cancer initiation and progression. Here, we show that silencing p53 expression with retrovirus-mediated small interfering RNA can delay the senescence and extend cell passage number, but is not sufficient to immortalize normal ovarian surface epithelial cells. Introduction of the catalytic subunit of telomerase is similarly insufficient to achieve immortalization. However, concurrent disruption of p53 expression with small interfering RNA retroviral constructs and ectopic expression of the catalytic subunit of telomerase was sufficient to induce cellular immortalization in 3 of 3 human ovarian surface epithelial cell cultures tested. The immortalization is associated with increased telomerase activity and telomere length, and attenuated response of cell-cycle regulatory proteins to irradiation. The resultant immortal cells continued to express the same specific cytokeratins 8 and 18 as parental cells did, indicating that the epithelial characters are still maintained in the immortal cells. In addition, the immortalized cells are non-tumorigenic and nearly diploid, which is in constrast with one immortalized by SV40 T/t antigens and hTERT. As both p53 pathway dysfunction and activation of telomerase are commonly present in human ovarian cancer, these immortal cells provide an authetic cell model system for the study of the human ovarian cancer initiation, progression, differentiation and chemoprevention.     

Human papillomavirus type 16 E6 and E7 genotypes in head-and-neck carcinomas

Human papillomavirus type 16 (HPV16) is associated with squamous cell carcinomas of the head and neck (HNSCC) particularly from the Waldeyer's tonsillar ring. A causal role of HPV16 in carcinogenesis is linked to the activity of the viral oncoproteins E6 and E7 which inactivate the cellular tumor suppressors p53 and pRB, respectively. Lack of E6 expression in HPV16-positive HNSCC has been reported, in some cases caused by disruption of the E6 gene. We have examined the status of the HPV16 E6-E7 gene region in tumor and metastasis samples of 24 HNSCC patients employing genomic PCR. No cases with a disrupted E6-E7 region could be identified. Sequence analysis of the E6-E7 segments revealed three different HPV16 E6-E7 genotypes: the HPV16 prototype (6 of 21 cases), the E6 variant T350G (8 of 21 cases), and the E6-E7 variant A131G/C712A (7 of 21 cases). The E6 variants T350G and A131G have been associated with increased oncogenic potential in cervical cancer patients depending on host genetic factors. Their high prevalence in the HNSCC samples studied indicates that they may be important also in HNSCC development.