Biomedical subjects

V Defendi

Publications and source records attributed to V Defendi.

At least 19 recordsLinked to original sources

PubMed1993

Genetic analysis of in vitro progression of human papillomavirus-transfected human cervical 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.

Base Sequence↗

PubMed1993

The state of p53 in primary human cervical carcinomas and its effects in human papillomavirus-immortalized human cervical cells.

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.

Base Sequence↗

PubMed1992

Transformation by human papillomavirus type 16 (HPV16) DNA but not HPV6b DNA is enhanced by addition of the human cytomegalovirus enhancer.

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.

Animals↗

PubMed1992

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.

3T3 Cells↗

PubMed1991

Evolution of in vitro transformation and tumorigenesis of HPV16 and HPV18 immortalized primary cervical epithelial cells.

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.

Cell Transformation, Neoplastic↗

PubMed1990

Human papillomavirus type 6b DNA required for initiation but not maintenance of transformation of C127 mouse cells.

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.

Animals↗

PubMed1989

Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes.

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.

Base Sequence↗

PubMed1989

Differential effects of human papillomavirus type 6, 16, and 18 DNAs on immortalization and transformation of human cervical epithelial cells.

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.

Adult↗

PubMed1988

Transformation of C127 mouse fibroblasts by human papillomavirus 16.

We have compared the ability of cloned DNAs of HPV16, a human papillomavirus associated with cervical carcinoma, and BPV1, a papillomavirus inducing skin lesions in cattle, to transform murine C127 cells. Unlike BPV1, HPV16 DNA failed to induce foci when C127 cells were transfected and maintained as monolayers; HPV16-transformed C127 cells could only be detected after cotransfection with HPV16 and pSV2neo DNA, selection for resistance to G418, and assay of pooled selectants for colony growth in agar. HPV16 and BPV1 C127 cells differed in terms of the size and morphology of their colonies in agar, but not in their colony-forming efficiencies. In addition, the tumors they induced in nude mice were clearly histologically distinct, with the HPV16 C127 tumors considerably more anaplastic. The HPV16 C127 cells contained viral DNA at high copy numbers integrated at random sites in the C127 genome, while the BPV1 C127, as expected, contained episomal BPV1 DNA molecules. The high complexity of the integrated HPV16 DNA was maintained in the pooled cells grown through extended passage in vitro, in clonal lines derived from single agar colonies, in nude mouse tumors induced by the cells, and in a nude mouse tumor-derived cell line, indicating the stability of the HPV16 sequences in the cells. HPV16 transcripts in the transformed C127 cells were present in three size classes (1.5, 2, and 4 kilobases) on Northern blots. The different transformed phenotypes in the same cell line induced by two structurally similar, yet distinct viruses imply differences in the underlying transforming mechanisms and possible different virus-host cell molecular interactions.

Animals↗

PubMed1988

A viral-cellular junction fragment from a human papillomavirus type 16-positive tumor is competent in transformation of NIH 3T3 cells.

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.

Animals↗

PubMed1987

DNA-binding activity of papillomavirus proteins.

We demonstrate DNA binding by papillomavirus (PV) open reading frame (ORF) proteins that correspond to the early transforming and trans-activating (E6 and E2) and late structural regions (L2 and L1) from bovine PV type 1 and human PV types 6b and 16. All PV proteins were synthesized in Escherichia coli and had a common 13-amino-acid leader sequence from the expression vector pRA10. Antibodies have been generated in rabbits against these PV proteins. The PV ORF proteins bind double-stranded DNA, and this activity is demonstrated to be inherent to the PV proteins. DNA-binding activity by PV proteins is optimal at 50 mM NaCl and at pH 7.0. For some PV proteins (e.g., bovine PV type 1 E2), DNA binding is enhanced at a lower pH (pH 6.0) and NaCl concentration (50 to 100 mM). DNA binding is inhibited by the appropriate antibodies. The possible significance of these findings is discussed in relation to the genetic and structural evidence on the function of these ORFs.

Amino Acid Sequence↗

PubMed1986

Expression of the E2 open reading frame of papilloma viruses BPV1 and HPV6b in Escherichia coli.

A new expression vector, pRA10, has been constructed for the expression of the open reading frames (ORF) of bovine (B) and human (H) papilloma viruses (PV). This vector is a derivative of pAJ pi and contains 15 restriction sites proximal to the lambda PL promoter, offering considerable versatility for insertion of different ORFs. This vector was used specifically to express the E2 ORF gene products from BPV1 and HPV6b at high level in Escherichia coli. The genuine nature of these proteins was demonstrated by restriction map analysis of expression vector plasmids to insure proper orientation, nucleotide sequence analysis to demonstrate in-frame insertion, E2 ORF protein production by expression-vector plasmids, and not by appropriate controls and, immunoprecipitation of E2 proteins by antibody specific for a common N-terminal sequence derived from the expression vector.

Animals↗

PubMed1985

Changes in fibronectin synthesis and binding distribution in SV40-transformed human keratinocytes.

We have studied the synthesis and distribution of fibronectin in human epidermal keratinocytes infected by SV40, a system in which the acquisition of transformed properties occurs in a sequential and progressive manner. Immunofluorescence studies showed that cultured uninfected keratinocytes do not exhibit fibronectin on the superficial cell surface, but that virus-infected cells come to display superficial fibronectin-containing cables in a density-dependent manner after a certain point in the transformation process. In contrast, organized arrays of fibronectin-containing fibrils associated with the cell-substrate attachment complex were seen in uninfected keratinocytes and in virus-infected cells at all stages of the transformation process. Studies of fibronectin synthesis using metabolic labelling of cell proteins with 35S methionine showed that viral infection caused a striking increase in overall fibronectin synthesis, although with a much higher proportion of newly synthesized fibronectin being secreted into the cell culture medium than in the case of the uninfected cells.

Cell Line↗

PubMed1985

Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40.

Transformation of human epidermal keratinocytes by the oncogenic virus SV40 is a stage-specific process in which normal patterns of differentiation are progressively altered over time following infection. Within the context of this scheme, we examined the keratins produced by the infected cells. Immunofluorescence studies indicated that viral infection led to the formation of variant cells visibly lacking the normal keratin cytoskeleton after about 10-15 serial passages (60-90 cell generations) post infection. Analyses of variant cell formation in clonal populations grown on palladium islands revealed that the variants were derived within 2-3 cell divisions from cells containing an apparently normal keratin cytoskeleton, but that variant formation depended upon cell density. Immunoprecipitation of 35S-methionine labelled keratins from the infected keratinocytes revealed a gradual loss of the normal 46, 50, 56 and 58Kd keratin species over a period of many months after infection. The loss of the normal keratins was accompanied by the appearance of at least two species in the 48-52Kd size range not present in uninfected cells and the enhancement of a third, 40Kd, protein quite early after infection. Analysis of the altered keratin patterns on two-dimensional acrylamide gels using either isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHG) along the first dimension showed that the infected cells produced basic keratins which increased in relative abundance as cells became more transformed with serial passage including at least five isoelectric forms not seen in uninfected cells. Translation of poly A+ RNAs from the infected cells indicated that the altered keratin synthesis probably reflects changes in the translatable mRNA pool.

Cell Count↗

PubMed1985

Keratin gene expression in simian virus 40-transformed human keratinocytes.

Previous reports from this laboratory indicate that cultured simian virus 40 (SV40)-transformed human keratinocytes express keratin proteins characteristic of simple epithelia that are not found in their untransformed counterparts. In this study we show by in vitro translation and RNA transfer blot analysis that the altered keratin synthesis reflects changes in the abundance of specific keratin mRNAs. SV40-transformed keratinocytes have a reduced abundance of transcripts for 58-, 56-, 52-, 50-, 48-, and 46-kDa keratin species, compared with uninfected cultured keratinocytes, but express significant levels of transcripts for 52-, 45-, and 40-kDa keratins, typical of simple epithelia. The SV40-transformed cells also express mRNA for a 48-kDa keratin that is unique to SV40-transformed keratinocytes. Analysis of the keratin genome with keratin-specific cDNAs as probes indicates that the changes in keratin transcription are not correlated with gross rearrangements of the keratin genome. These results suggest that analysis of viral transformation of cultured keratinocytes affords a novel approach to study mechanisms regulating keratin gene expression.

Cell Transformation, Viral↗

PubMed1984

Re-expression of differentiated properties in SV40-infected human epidermal keratinocytes induced by 5-azacytidine.

Infection of human epidermal keratinocytes by the oncogenic virus SV40 leads to progressive inhibition of the normal differentiation process in vitro. Treatment of infected cells with 5-azacytidine (5-aza-CR) over a 24-h period produced a striking enlargement and pronounced flattening of cells within 5-7 days following removal of the agent. This morphological change was accompanied by a several-fold increase in the number of cells staining positively for the cell envelope precursor protein, involucrin, and in the exfoliation of cornified envelope bearing cells from the monolayer. The drug-treated cultures at high passage levels were stained by immunofluorescence using monoclonal antibodies to keratin classes associated with different epidermal layers. These experiments revealed that 5-aza-CR caused the re-expression of two keratin classes (suprabasal and stratum corneum-associated), whose synthesis had been suppressed during the transformation process. 5-Aza-CR also brought about re-expression of 58 and 56 kD keratin markers of epithelial keratinization and stratification, as well as of 40 and 49-52 kD keratin markers of viral transformation. However, the responsiveness to the drug was gradually lost over time following infection.

Azacitidine↗

PubMed1984

Simple epithelial nature of some simian virus-40-transformed human epidermal keratinocytes.

Previous studies have indicated that some Simian-virus-40-transformed human epidermal keratinocytes (SV40-HE) undergo significant changes in their growth and differentiated properties. To better understand the significance of these changes, we have characterized the keratins of SV40-HE cells by one- and two-dimensional immunoblot analysis using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that our SV40-HE cells have lost the Mr 58,000 (No. 5), Mr 56,000 (No. 6), Mr 50,000 (No. 14/15), Mr 48,000 (No. 16), and Mr 46,000 (No. 17) keratins that are expressed by cultured normal human keratinocytes. Instead, these cells express mainly Mr 52,000 (No. 8), Mr 45,000 (No. 18), and Mr 40,000 (No. 19) keratins, a set highly characteristic of simple epithelial cells. Furthermore, our SV40-HE cells have ceased to express involucrin, another marker for keratinocytes, and have a greatly diminished ability to undergo in vitro stratification. These results suggest that epidermal cells can sometimes lose their keratinocyte features as a consequence of viral transformation. This finding may have important implications regarding the mechanisms of epithelial differentiation and tumorigenesis and in the use of keratinocyte markers for tumor diagnosis.

Cell Differentiation↗

PubMed1984

Structure of simian virus 40 DNA in primary and derived mouse tumors.

Primary tumors induced by Simian Virus 40 (SV40) in nude mice, lines derived from them, and subsequent transplanted tumors were analyzed as to the state of the viral DNA. It was found that SV40 DNA is present only as integrated molecules, each tumor having a different integration pattern; all tumors were SV40 T-antigen positive by immunofluorescence. One tumor and its derivatives in vitro and in vivo have maintained a single integration structure consisting of a head-to-tail arrangement of a molecule slightly larger than whole-length viral DNA (approximately equal to 1.1). This tumor expresses a full-sized T-antigen and a Mr 54,000 host-associated protein.

Animals↗