Mary M. Pater

Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix.

A total of eight human epithelial cell lines derived from the carcinoma of the cervix were examined for the presence of human papillomaviruses (HPVs) types 16 and 18 DNA sequences. Six out of eight cell lines contain sequences hybridizing to the DNA of these viruses. Two of the cell lines contain sequences hybridizing specifically to HPV 16. One of these two cell lines contains all of the HPV 16 sequences and the other cell line is missing fragments containing early regions E2 and E4 and some of the late regions. Four of the cell lines contain sequences hybridizing specifically to HPV 18. All these cell lines are missing fragments containing early regions E2, E4, and E5. Interestingly, all the cell lines contain sequences corresponding to early regions E1, E6, and E7.

Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells

Previously, a Bcl-2-interacting protein, BAG-1, was cloned from mouse cells and was shown to interact with several other proteins and to be important for inhibition of apoptosis. Human BAG-1 (hBAG-1) cDNA, recently isolated by us and two other groups, has been shown to be identical to a hormone receptor-binding protein, RAP46. However, different molecular masses of hBAG-1 protein products were noted by these three groups. Here we demonstrated that hBAG-1 protein was expressed as four isoforms, designated p50, p46, p33 and p29, with apparent molecular masses of 50 kDa, 46 kDa, 33 kDa and 29 kDa, respectively. Deletion, site-directed mutagenesis and in vitro transcription/translation analysis showed that the four protein products of hBAG-1 were expressed by alternative initiation from four different start codons through a leaky scanning mechanism. Furthermore, we demonstrated that the distinct forms of hBAG-1 have different subcellular localizations, suggesting that they may have distinct functions in the cells. Characterization of hBAG-1 RNA and protein also showed that hBAG-1 was overexpressed in human cervical, breast and lung cancer cell lines. Taken together, these data clarify the conflicting observations reported in the literature and suggest that hBAG-1 is expressed as four forms of protein products, which may play a differential role in apoptosis and oncogenesis of human cells.

Oncogenic transformation by human papillomavirus type 16 deoxyribonucleic acid in the presence of progesterone or progestins from oral contraceptives

Compelling evidence supports a role of certain types of human papillomaviruses as the cause of cervical cancer. In addition to human papillomaviruses, other agents, such as hormones, have been implicated as cofactors in this type of neoplasia. In this study we provide evidence for oncogenic transformation of primary baby rat kidney cells by human papillomavirus type 16 deoxyribonucleic acid plus ras oncogene in the presence of progesterone but not estrogen. Integrated and intact human papillomavirus type 16 deoxyribonucleic acid is present and expressed in all the five progesterone-transformed colonies that we examined. Moreover, all these cell lines are capable of anchorage-independent growth and induce tumors in syngeneic animals. We also observed oncogenic transformation with human papillomavirus type 16 deoxyribonucleic acid plus ras in the presence of ethanol-soluble extracts from two brands of commonly used oral contraceptive tablets. No transformation is achieved in the presence of ethanol-soluble extracts from the inert tablets, provided in packages of each brand of oral contraceptive. These results may have implications for a papillomavirus-hormone link to cervical neoplasia.

Role of steroid hormones in potentiating transformation of cervical cells by human papillomaviruses

Human papillomaviruses (HPVs) are etiologically involved in cervical neoplasia, and epidemiological evidence suggests that steroid hormones can increase the risk of this cancer in HPV-infected women. Steroids can interact with hormone-response elements in the viral long control region, enhancing HPV transcription and resulting in transformation of cervical cells. Subsequent malignant progression may involve virus-induced chromosomal instability, facilitating viral DNA integration and deregulation of gene expression.

Human papillomavirus types 16 and 18 sequences in early cervical neoplasia

A total of 100 colposcopic biopsies from patients with abnormal Papanicolaus tests were surveyed for the presence of human papillomavirus (HPV) types 16 and 18 sequences by spot-blot hybridization. HPV 16 and 18 DNA sequences were detected in 58% of the biopsies. None of the cervical intraepithelial neoplasia grade I (CIN I) contained HPV 16 while 50% of the CIN III lesions (carcinoma in situ, CIS) contained HPV 16. HPV 18-related sequences were equally represented in CIN I, II, and III. Southern-blot hybridization of total undigested cellular DNA revealed the presence of HPV DNA sequences only in an episomal form. While the restriction enzyme patterns in HPV 16-positive samples were mostly identical to the originally cloned sequence, the restriction enzyme pattern for HPV 18-positive samples were different from that of HPV 18 but identical to each other. Furthermore, this DNA hybridized more strongly to HPV 18 under nonstringent conditions, suggesting a new type.

Malignant transformation of HPV 16-immortalized human endocervical cells by cigarette smoke condensate and characterization of multistage carcinogenesis.

A number of epidemiological studies indicate that cigarette smokers are at increased risk of developing cervical cancer. However, convincing biological evidence is lacking. This report examines the biological and cellular role of human papillomavirus (HPV) type 16 and cigarette smoke in multistage cervical carcinogenesis. Two lines of HPV16‐immortalized human endocervical cells (HEN‐16 and HEN‐16‐2) generated from primary cells (HEN) were treated with cigarette smoke condensate (CSC). CSC‐treated, but not untreated, HEN‐16 and HEN‐16‐2 formed tumors that were invasive squamous cell carcinomas in nude mice. The tumors were used to initiate 2 tumor lines of cells (HEN‐16T and HEN‐16‐2T, respectively). Cells of both tumor lines, compared with HEN, HEN‐16 and HEN‐16‐2, featured: (a) tumorigenicity, (b) distinct morphologies in monolayer and organotypic (raft) cultures, (c) faster growth in serum plus high calcium levels after immortalization and after transformation, (d) higher saturation density and (e) anchorage‐independent growth. Our results provide unique direct in vitro evidence that cigarette smoke causes cancer in HPV‐containing cervices.

Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells

A series of recombinant plasmids carrying various DNA fragments of SV40 early region were used to test for their ability to immortalize primary cultures of rat embryo (RE) and human embryonic kidney (HEK) cells. When primary RE cells were transfected with plasmids containing an entire early region of wild-type SV40- or a deletion mutant in the small tumor (t) antigen, dl 1410-DNA, they were all immortalized. The immortalized cells could grow in soft-agar medium and produced large tumor (T)-antigen. Cultured RE cells transfected with pW2-t, which contains a deletion in the large-T-specific coding region, also gave rise to continuous cell lines. Interestingly, two of nine RE lines immortalized by pW2-t could also grow in soft-agar medium. The plasmid pW-t8 carrying a similar fragment of SV40 DNA as pW2-t, but lacking the processing and polyadenylation signal sequences, also immortalized RE cells. Surprisingly, the plasmid pD-t1 which contains neither the intact large-T nor the small-t function also immortalized RE cells. However, the RE lines immortalized by pW-t8 or pD-t1 were unable to grow in soft-agar medium and displayed a wide range of growth phenotypes. On the contrary, when primary HEK cells were used for immortalization experiments, only those SV40 plasmids carrying the intact large-T function were able to generate immortalized lines. The growth properties of these immortalized HEK lines can be categorized into two groups. Those HEK lines immortalized by the large T alone grew slightly denser and rounder than their parental normal HEK cells, while those immortalized by both the large-T and small-t antigens grew extremely fast, reached higher density, piled up on each other, and were anchorage independent. In addition, when these SV40 plasmids were used to directly transform primary HEK cells by the focus assay, the large-T clone, pD3-05, only transformed HEK cells to form light foci. Transfection by the large-T plus the small-t sequences either in cis or in trans, did increase the frequency of focus formation, and gave rise to dense foci which could grow in soft-agar medium.

Transformation by purified early genes of simian virus 40

A rapid soft-agar assay using baby hamster kidney (BHK21 cl.13) cells has been developed to establish the functional roles for the large T and small t antigens of SV40 in transformation. Plasmids expressing either large T or small t antigens of SV40 have also been constructed and these plasmids have been used separately or in combination for transformation. A large T clone, pD3-05, containing a deletion in the small t-specific coding region [0.584-0.54 map units (mu)], transformed a low-background subclone of baby hamster kidney (BHK21 cl.13) cell line and F111 rat fibroblasts to anchorage independence at a low level (10-20 and 1%, respectively, of an early region clone from wild type [WT], pW2). A WT-derived small t clone, pW2-t, containing a deletion in the large T-specific coding region (0.373-0.169 mu), did not transform F111 cells, but transformed BHK21 cells at a very low level (about 2% of pW2). Another WT-derived small t clone, pW2-t/B1, containing a larger deletion in the large T-specific coding region (0.512-0.169 mu), did not transform either BHK21 or F111 cells. However, cotransformation with pD3-05 clone and pW2-t or pW2-t/B1 clone increased the frequency of transformation to about the same level as that of pW2. The ability of the small t clones to enhance the transformation efficiency of the large T clone was not due to recombination between the two plasmids, since cotransformation with pD3-05 and a small t clone without the polyadenylation [poly(A)] signal sequence from WT, pW-t8, did not increase the frequency of transformation. When the frequency of transformation was determined by the focus assay using F111 cells, pD3-05 transformed as well as pW2. Also, cotransformation with pD3-05 and pW2-t/B1 did not increase the frequency of focus formation. Therefore, the small t antigen was not required for this morphological transformation.

Identification and characterization of a JC virus pentanucleotide repeat element binding protein: Cellular nucleic acid binding protein

The JC virus (JCV) control region contains AGGGAAGGGA, the tandem pentanucleotide repeat element (Pnt2). Several proteins specifically interacted via Pnt2 to regulate the expression of JCV early promoter-enhancer (JCV(E)) or late promoter-enhancer (JCV(L)). In this study, a JCV Pnt2 oligonucleotide probe was used to screen a cDNA expression library from glial P19 mouse embryonal carcinoma cells. A cDNA clone was isolated by Southwestern blot assay and it produced a protein that reproducibly and specifically bound to Pnt2. This cDNA had 100% homology to one of three previously identified mouse cDNAs called cellular nucleic acid binding proteins (Cnbps). Cnbps are a highly homologous family of eukaryotic genes implicated in functional interactions with cytoplasmic RNA and regulatory DNA elements. An mRNA of 2.2 kb of Pnt2-interacting Cnbp (PCnbp) was seen in undifferentiated, muscle or glial P19 cells. When expressed from a cDNA expression vector as a fusion protein that also contained 115 kDa from beta-galactosidase, a Pnt2 binding protein (PCNBP) specifically bound to Pnt2 in Southwestern blots as a 30 kDa component of the 145 kDa fusion protein. Furthermore, JCV(E) expression was negatively regulated by PCnbp produced in vivo from the cDNA expression vector. Regulation of JCV(L) was unaffected. We suggest a novel role for CNBP as a PCNBP that interacts with Pnt2 in the negative transcriptional regulation of JCV(E).

Enhanced expression of anti-apoptotic proteins in human papillomavirus-immortalized and cigarette smoke condensate-transformed human endocervical cells: correlation with resistance to apoptosis induced by DNA damage.

Apoptosis plays an important role in various biological processes including embryogenesis, differentiation, homeostasis, and oncogenesis. We have developed a system composed of primary human endocervical cells (HEN), HEN immortalized by human papillomavirus (HPV) type 16, and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). To understand the role of apoptosis in the multistep oncogenesis of human cervical cells, we examined the expression of apoptosis‐associated proteins in our in vitro model system. The results showed no significant difference in the levels of apoptosis‐inducing proteins bak and bax among all the cell types examined. On the other hand, the levels of apoptosis‐inhibiting proteins bcl‐2, bcl‐xL and BAG‐1 increased progressively after immortalization and transformation. The p53 protein level decreased in the HPV16‐immortalized HEN and increased in one of two lines of the CSC‐transformed HEN. Further, the increased levels of apoptosis‐inhibiting proteins in the HPV16‐immortalized and the CSC‐transformed HEN correlated with progressively increased resistance of these cells to apoptosis induced by staurosporine or cisplatin. This study provided the first evidence that overexpression of apoptosis‐inhibiting proteins is important for both multistep oncogenesis and resistance of human endocervical cells to apoptosis induced by DNA‐damaging reagents. Mol. Carcinog. 22:95–101, 1998.