Faculty Bibliography

Wild-type (wt) p53 acts as a tumor suppressor, while certain mutant type (mt) p53 may exhibit 'oncogenic' function. We have recently demonstrated that human papillomavirus type 18 (HPV-18) E6 can partially overcome the growth-suppressive effects of wt p53, but it remains unclear what role p53 plays in cervical carcinogenesis. In this report, we have examined nine HPV-immortalized human cervical epithelial cell lines and 13 HPV-positive and two HPV-negative primary cervical cancers for p53 mutations by polymerase chain reaction--single-strand conformation polymorphism (PCR-SSCP). None of them contained p53 mutations in exons 5-9 where most p53 mutations in human tumors have been found. The entire p53-coding region of the two HPV-negative cervical cancers was sequenced and no mutations were noted. In order to examine the effects of wt p53 and mt p53 on HPV-immortalized human cells, we transfected HPV-immortalized cell lines with wt p53 and a mt p53 (mtp53Val-135). The results indicate that HPV-immortalized cells cannot tolerate large amounts of exogenous wt p53, while mt p53Val-135 can enhance transformation of these cells. The results support the notion that inactivation of wt p53 by E6 may be important for HPV-associated transformation and also suggests that mt p53 can act as an oncogene in HPV-immoralized human cells

The authors have established an in vitro model system which demonstrates the progression of the transformed phenotypes of human cervical epithelial cells transfected with human papillomavirus (HPV) type 16 and 18 DNAs. Both viral DNAs exhibit immortalizing potential; however, only HPV 18-immortalized cell lines progress to exhibit anchorage-independent growth and, in a limited number of cases, tumorigenesis. In this paper, the authors have examined the genetic basis for this in vitro progression step by step, including immortalization, anchorage-independent growth, and tumorigenicity of the HPV-transfected human cervical epithelial cells by cell fusion. The results suggest that (a) all three transformed phenotypes, i.e., immortalization, anchorage-independent growth, and tumorigenesis, in this in vitro cervical carcinogenesis model are a result of recessive changes in genes or processes involved; (b) inactivation of p53 and retinoblastoma protein is not sufficient for immortalization of human cervical epithelial cells; (c) HPV expression per se does not account for immortalization of human cervical epithelial cells; (d) immortalization of human cervical epithelial cells initiated by HPV can occur through different processes, although one of them is the most preferred; and (e) probably only one group of recessive genes appears to be involved in the loss of anchorage-dependent growth for HPV-immortalized human cervical cells

Primary human cervical epithelial cells immortalized by human papillomavirus type 16 (HPV16) DNA exhibit altered morphology and differentiation characteristic of transformation, but show a lack of transformed phenotype relative to HPV18 DNA immortalized cells in terms of anchorage-independent growth (Pecoraro, Lee, Morgan, and Defendi, 1991, Am. J. Pathol. 138, 1-8). This is completely corrected by inserting a strong heterologous enhancer derived from human cytomegalovirus DNA upstream from the HPV16 long control region. The cells immortalized by this DNA form colonies in agar comparable to those formed by HPV18 DNA immortalized cells. The enhanced transformation capability correlates with increased levels of HPV16 E6-E7 and E5 transcripts. The HPV16 DNA containing this strong enhancer also transforms C127 mouse cells with increased efficiency and strength relative to the natural HPV16 DNA, as measured by the numbers and size of the colonies in agar. The positive effects of this strong enhancer appear specific for HPVs associated with genital malignancies such as HPV16, since HPV6b DNA (primarily in benign tumors) with or without the strong cytomegalovirus enhancer is incapable of immortalizing primary human cervical epithelial cells or allowing efficient growth of C127 mouse cells in agar. These results suggest that the diminished oncogenic properties of HPV16 versus HPV18 DNA in cultured cells and in human malignancies may reside in the long control regions of these viruses and, additionally, may define another difference in the oncogenic properties of HPVs associated with benign or malignant genital neoplasia

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

Cervical carcinoma develops through a progressive spectrum of premalignant intraepithelial lesions (CIN I-III), the majority of which are associated with human papillomavirus (HPV) types 16 and 18. We established HPV16 and HPV18 immortalized human cervical epithelial cell lines and used them as a model to investigate the genesis and progression of cervical malignancy. The cell lines when cultured in vitro in a system mimicking their in vivo environment exhibit cytologic atypia and a variety of defects in morphologic differentiation at early passage compared to their normal counterparts. With increased passage, these alterations progress to more severe grades, histologically similar to CIN III; however only a limited number of the cell lines are tumorigenic, mimicking the epidemiologic evidence on the rate of conversion from premalignant to invasive carcinoma. The observed changes are not associated with alterations of viral DNA integration or expression and may reflect specific cellular events or changes in virus-host interactions associated with malignant progression

We describe the transformation of C127 mouse fibroblasts with human papillomavirus type 6b (HPV-6b) DNA, which is associated primarily with benign tumors of the human genital tract. The major transformed phenotype of the HPV-6b-transfected cells lines, which had been G418 selected, pooled, and maintained without subsequent selection, was tumorigenicity in nude mice. We found that, unlike that reported for other HPVs or papovaviruses, the transformed phenotype was expressed after a delay, in which the cells had undergone extensive culture passages (about 20 passages or 100 generations). Interestingly, the HPV-6b DNA had become reduced or nondetectable in copy number in the cells by the time the transformed phenotype was expressed and in most of the tumors induced by the cells in nude mice, indicating that high levels of HPV-6b DNA were not required for maintenance of the transformed phenotype. Clonal cell lines gave similar results. When continued G418 selection was used to maintain high-copy-number HPV-6b DNA, the cells were tumorigenic, indicating that high levels of HPV-6b DNA did not suppress tumorigenesis. These studies suggest that HPV-6b DNA initiates transformation of C127 cells but is dispensable for expression or maintenance of the transformed phenotype. Transformation by HPV-6b DNA in vitro may provide insights into the HPV type-specific association with benign versus malignant lesions in vivo and may elucidate some of the oncogenic processes involved in tumor progression

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types

The human papillomaviruses (HPVs) are associated with specific benign and malignant lesions of the skin and mucosal epithelia. Cloned viral DNAs from HPV types 6b, 16, and 18 associated with different pathological manifestations of genital neoplasia in vivo were introduced into primary human cervical epithelial cells by electroporation. Cells transfected with HPV16 or HPV18 DNA acquired indefinite lifespans, distinct morphological alterations, and anchorage-independent growth (HPV18), and contain integrated transcriptionally active viral genomes. HPV6b or plasmid electroporated cells senesced at low passage. The alterations in growth and differentiation of the cells appear to reflect the progressive oncogenic processes that result in cervical carcinoma in vivo

A 4.4-kilobase DNA fragment (T4.4) from a human tumor (comprising part of the human papillomavirus type 16 E6 promoter; the E6, E7, and part of the E1 open reading frames; and cellular sequences) was found to be competent to fully transform NIH 3T3 cells. This competency resides in the whole hybrid DNA fragment, since the separate viral or cellular DNA sequences were not active. Abundant E6-E7 transcripts were found in the transformed cells. When the cellular fragments were substituted with polyadenylation sequences from polyomavirus or simian virus 40 DNA, little or no restoration of transforming activity was observed. In experiments in which an exogenous reporting gene, that for chloramphenicol acetyltransferase, was used, the possibility was excluded that the cellular flanking sequences act as a traditional enhancer; yet, when the cellular sequences were placed downstream of a chloramphenicol acetyltransferase expression vector (pSV2 CAT), activity of the reference gene was clearly enhanced. These results indicate that DNA containing human papillomavirus type 16 open reading frames E6 and E7 isolated from the genome of a human tumor has transforming potential, that this potential is realized when the viral DNA is joined to cellular sequences, and that the cellular sequences function in a more complex way than by simply providing polyadenylation signals