Transformation of Bloom's syndrome fibroblasts by DNA transfection.

Nonmalignant diploid human fibroblast cells (GM3498B) derived from a skin biopsy of a patient with Bloom's syndrome have been transformed by transfection with DNA from a tumorigenic mouse cell line (Ha-8) carrying a single copy of the Harvey murine sarcoma virus (Ha-MuSV) genome. The transformed cell lines have an extended life-span, form colonies in agarose, and proliferate in nude mice--characteristics of neoplastic transformation. Like the parental cells, they also exhibit a high spontaneous level of sister chromatid exchanges. Finally, the transformed cells contain most, if not all, of the Ha-MuSV genome as well as the human rasH sequence. These experiments show that these diploid nonmalignant human cells can be used as recipients in transfection experiments for studying the genetic control of neoplastic transformation.

A transforming ras gene in tumorigenic guinea pig cell lines initiated by diverse chemical carcinogens.

Fetal guinea pig cells were transformed by treatment with four different chemical carcinogens including nitroso compounds and polycyclic hydrocarbons. As a consequence of this treatment, oncogenes capable of transforming NIH/3T3 cells became activated in each of five independently established clonal guinea pig cell lines. Molecular characterization of representative NIH/3T3 transformants revealed that the same oncogene was present in each of the cell lines tested. Moreover, detection of this transforming gene paralleled the acquisition of tumorigenic properties by these neoplastic cells.

Differential early viral gene expression in two stages of human papillomavirus type 16 DNA-induced malignant transformation.

Human papillomavirus (HPV) type 16 DNA induces progressive transformation in NIH 3T3 cells. Two types of cell lines, PM3T3G0 and PM3T3Fo, were isolated by G418 or focus selection, respectively, after transfection of cells by a recombinant HPV 16 DNA carrying the neo gene. These cell lines exhibited distinct phenotypes compared with controls. Saturation densities of PM3T3G0 and PM3T3Fo lines were two- to three- and five- to sevenfold greater than that of control NIH 3T3 cells, respectively. Neither cell type required high serum for growth, in contrast to NIH 3T3 cells. PM3T3G0 lines were premalignant, whereas PM3T3Fo lines manifested tumorigenicity within 2 weeks. Subpopulations of three PM3T3G0 lines underwent progressive transformation as reflected by focus formation. Analysis of HPV 16-specific mRNA species demonstrated that high levels of early and late gene expression were detected in premalignant PM3T3G0 lines, whereas relatively low quantities of selected gene messages were expressed in malignant transformants. Thus, high levels of viral gene expression are not crucial for malignant transformation.

Neoplastic transformation of Syrian hamster cells by putative epoxide metabolites of commercially utilized chloroalkenes.

The transforming ability of six epoxides of structurally related chloroalkenes was determined with a quantitative Syrian hamster embryo cell model. The epoxides used were cis-1-chloropropene oxide (c-CPO), trans-1-chloropropene oxide (t-CPO), cis-1,3-dichloropropene oxide, trans-1,3-dichloropropene oxide, trichloroethylene oxide (TCEO), and tetrachloroethylene oxide (PCEO). All six epoxides induced morphologic transformation of Syrian hamster embryo cells and caused cell lethality as reflected in the reduced cloning efficiency; in all instances, transformation was observed with less than 25% toxicity. The potency of the various epoxides was influenced by the difference in stability of the compounds. The results with c-CPO, t-CPO, TCEO, and PCEO were consistent with a linear dose response. The transformation results reflect the carcinogenicity of the parental chloroalkenes tested thus far. Furthermore, if the epoxides are metabolic intermediates of the chloropropene parent chloropropenes, the epoxides are probably proximate carcinogens.

Excision and postreplication DNA repair capacities, enhanced transformation, and survival of Syrian hamster embryo cells irradiated by ultraviolet light.

The frequency of ultraviolet light (UV)-induced neoplastic transformation of Syrian hamster embryo cells (HEC) is enhanced 3- to 10-fold when the cells are first treated with either X-irradiation or with methyl methanesulfonate. Maximum enhancement occurs when the interval between the two treatments is 48 hr. The relevance of UV-induced transformation to neoplasia is confirmed because the transformation to neoplasia is confirmed because the transformants produce tumors when injected into nude mice. Excision and postreplication DNA repair were studied to determine whether the enhanced transformations were associated with either of these repair mechanisms. Independent of X-ray or of methyl methanesulfonate pretreatment, approximately 25% of the pyrimidine dimers are excised within 24 hr in cells irradiated with UV with 3 J/sq m. During this period, more than 70% of the genome of cells irradiated with UV has been replicated. Postreplication repair is measured by the time required to chase pulse-labeled nascent DNA strands to parental-sided DNA. Regardless of pretreatment, 1 and 3 hr are required for pluse-labeled DNA in control and irradiated (10 J/sq m) cells, respectively, to reach parental size. Therefore, no correlation is found between a change in the rate of excision or postreplication repair and enhancement of transformation. Relative to control cloning efficiency, the survival of HEC contain more than 10(5) pyrimidine dimers/genome. The level of survival is similar to the survival of human skin fibroblasts which excise pyrimidine dimers four to five times as efficiently. Moreover, postreplication repair cannot account for the ability of these cells to survive because it is three times slower than in human fibroblasts. Therefore, other repair mechanisms must be responsible for HEC survival and transformation.

Human fibroblast strain with normal survival but abnormal postreplication repair after ultraviolet light irradiation.

Postreplication repair has been studied in ultraviolet light (UV)-irradiated fibroblast strains derived from eight apparently normal control donors and seven xeroderma pigmentosum patients. One control donor strain had an intermediate defect in postreplication repair similar to that in excision-deficient xeroderma pigmentosum fibroblasts. However, unlike the xeroderma pigmentosum strains, this control donor strain had normal UV-induced unscheduled DNA synthesis and normal survival after irradiation with UV. This unique fibroblast strain should after irradiation with UV. This unique fibroblast strain should be useful in studies designed to elucidate the possible role of postreplication repair in UV-induced carcinogenesis and mutagenesis.

Inhibition of radiation-initiated and -promoted transformation of Syrian hamster embryo cells by lymphotoxin.

Sensitivity of Syrian hamster cells to the anticarcinogenic action of hamster lymphotoxin obtained from mitogen-stimulated peritoneal lymphocytes depends on the stage of transformation, initiation, and promotion. Dose-response results with 12-O-tetradecanoylphorbol-13-acetate (TPA) plus X-irradiation parallel those obtained on mouse skin. Twice as much lymphotoxin was required to obtain a 50% reduction in TPA-promoted transformation as in nonpromoted transformation, demonstrating a difference in initiated and promoted cell sensitivity to lymphotoxin. In a study of promoted transformation, 48-hr lymphotoxin treatment before or immediately after X-irradiation, or during TPA exposure, caused a persistent inhibition independent of when lymphotoxin was added. The degree of sensitivity of different steps in carcinogenesis as the cells underwent the physiological changes associated with transformation was examined more precisely with 6-hr lymphotoxin treatments. Lymphotoxin treatment before irradiation and TPA caused a transient cellular change. When the cells were initiated within 2 days after lymphotoxin exposure, the induction of promoted transformation was inhibited. Results were similar with nonpromoted transformation. Lymphotoxin became a more effective anticarcinogen as the interval between the lymphotoxin pulse and carcinogen insult or TPA addition was reduced. When added during the last 6 hr of the experiment, lymphotoxin was equally inhibitory, whether or not TPA was present. Thus, lymphotoxin induces an anticarcinogenic physiological state that is short-lived or transient; the temporal relationship between lymphotoxin and carcinogen exposure is important for preventing initiated or promoted transformation.

Down-regulation of keratin 14 gene expression after v-Ha-ras transfection of human papillomavirus-immortalized human cervical epithelial cells.

Keratin expression in human cervical squamous cell carcinoma (SCC) lines differed significantly from both normal and human papillomavirus (HPV) immortalized exocervical cells. Keratin 14 (K14) expression, determined by protein synthesis and mRNA levels, was dramatically down-regulated in the cervical SCC lines while keratin 5 (K5) expression was not. K14 expression was similarly down-regulated in an HPV-16 immortalized cervical cell line after tumorigenic transformation with recombinant v-Ha-ras DNA. Cultures derived from nude mouse tumor explants also exhibited an altered keratin profile and the levels of K14 protein synthesis, as well as K14 mRNA, were not detectable. In both cases K5 protein synthesis was not significantly down-regulated. In addition, neoplastic cervical SCC lines exhibited up-regulation of keratins 7, 8, 13, and 19, combined with slight down-regulation of keratins 6 and 16. Epidermal keratinocytes responded in a different manner to exocervical cells. Transfection of human papillomavirus-immortalized epidermal keratinocytes with the BglII N fragment of herpes simplex virus 2 produced a neoplastic cell line, but K5 and K14 expression remained unchanged. Thus, neoplastic transformation of human exocervical cells, both in vivo (spontaneous cervical SCC) and in vitro (HPV-16- and v-Ha-ras-induced cervical SCC), is accompanied by characteristic changes in keratin expression. The specific down-regulation of K14 in these tumorigenic cervical cells, in the absence of significant changes in the expression of K5, implies that the normal coordinate regulation of K5 and K14 gene expression has been uncoupled.

Altered polypeptide expression associated with neoplastic transformation of Syrian hamster cells by bisulfite.

Neoplastic transformation of Syrian hamster fetal cells by bisulfite is associated with qualitative and quantitative polypeptide changes. Amino acid-labeled [14C]polypeptides from neoplastic and nontransformed parental fetal cells were separated by two-dimensional gel electrophoresis and analyzed by computerized microdensitometry of autoradiographic patterns. Approximately 1000 polypeptides from parental fibroblasts at population doublings ranging from 4 to 20, and those from colony-derived malignant cell lines were compared. Most were identical. Seven malignant lines exhibited 4 qualitative polypeptide changes: 2 polypeptides had shifted slightly to the acidic side, 1 new polypeptide was observed, and 1 polypeptide was absent. The transformed bisulfite lines differed quantitatively from control cells in that 10-25% and 2-4% of the polypeptides exhibited differences in expression greater than 2- and 4-fold, respectively. Furthermore, there were 21 specific polypeptides with coordinate quantitative changes in all transformed lines. Because bisulfite at neutral pH fails to induce any significant DNA changes at concentrations that cause transformation, polypeptides expressed immediately or 48 h after bisulfite treatment were compared to those of non-treated controls, and no differences were found. Even though bisulfite does not induce detectable DNA damage or early post-treatment changes in polypeptide expression, a consistent set of qualitative and quantitative changes were observed after transformation. The qualitative polypeptide changes found in the bisulfite-induced malignant lines were similar to those seen in a benzo(a)pyrene-induced malignant line. This suggests that there is a convergence of pathways responsible for carcinogenesis independent of the nature of initiation.

Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA.

An in vitro system for studying the interaction between human papillomavirus (HPV) 16 and 18 recombinant DNA and normal human exocervical epithelial cells is described. Eight HPV-immortalized human exocervical epithelial cell lines were established; all the lines contained either integrated HPV16 or 18 sequences and expressed HPV mRNAs. Thus, integration and expression appear to be required for immortalization. Immortalized cells (greater than 200 population doublings to date) divided rapidly (doubling time of 30 to 46 h) and morphologically resembled primary cultures of normal human exocervical epithelial cells. They expressed a keratin pattern consistent with their origin from exocervical epithelium. When cultured at high density or in the presence of serum they terminally differentiated. Sublines resistant to terminal differentiation were selected by growth in serum-supplemented medium. Keratin pattern changes suggest they have some properties in common with cervical squamous carcinoma cells. However, HPV-immortalized cell lines were not tumorgenic in nude mice. Thus, HPV16/18 is not carcinogenic by itself. These cell lines represent an appropriate model for studying factors that regulate HPV gene expression in normal cervical epithelial cells and examining the influence of cocarcinogens on neoplastic progression.

Frequent activation of non-ras transforming sequences in neoplastic Syrian hamster cells initiated with chemical carcinogens.

Carcinogen-caused transformation of Syrian hamster embryo cells has been widely used as a model for experimental carcinogenesis. However, analysis of the molecular mechanisms of hamster cell transformation has been limited. To expand the understanding of the molecular basis of this system, 22 independently derived Syrian hamster neoplastic cell lines initiated with chemical carcinogens were screened for the presence of dominant transforming sequences by DNA transfection into mouse NIH3T3 cells. High molecular weight DNAs from 12 (55%) of these cell lines transformed NIH3T3 cells through serial transfection cycles. NIH3T3 transformants contained hamster-specific repetitive sequences, which co-segregated with the transformed phenotype in successive transfection rounds. Results from Southern hybridization analyses and p21ras mobility assays indicated the presence of N-ras oncogenes, presumably activated by point mutations at codon 61, in 3 of the 12 (25%) transfection positive lines, all initiated with sodium bisulfite; non-ras transforming sequences were apparently activated in the remaining 9 (75%) lines. DNA prepared from NIH3T3 transformants derived from cell line 81C39 was analysed by Southern hybridization with a battery of 38 probes including non-ras oncogenes known to score as positive in the NIH3T3 assay as well as other retroviral and mammalian oncogenes. Each probe hybridized to DNA fragments showing the mobility characteristic of NIH3T3 protooncogenes, but failed to detect homolog sequences of hamster origin, even under hybridization conditions which allowed their detection in hamster DNA. Results show that ras activation occurs at a low frequency in hamster neoplastic transformation and strongly suggest that novel transforming sequences are activated, thus validating the use of this system for investigating the role of non-ras transforming sequences in neoplasia.

Induction of human cervical squamous cell carcinoma by sequential transfection with human papillomavirus 16 DNA and viral Harvey ras.

Clinical and epidemiological data are consistent with the hypothesis that human papillomaviruses (HPVs) are a factor in genital, particularly cervical cancer. Although HPV16 and 18 are found primarily in cervical malignancy, the transfection of HPV16 or 18 DNA into cervical cells results in immortalization but not tumorigenicity. The addition of activated Ha-ras, an oncogene found in some cervical cancers expressing HPV16 or 18, to HPV16-immortalized human cervical cells results in malignancy as proven by the formation of cystic squamous cell carcinomas by HPV16-Ha-ras cells in nude mice. This two-stage model utilizing relevant human cells demonstrates that HPVs play a critical role in cervical malignancy and provides a system for elucidating critical cellular changes associated with progression to malignancy.

Cell lines containing and expressing the human herpesvirus 6A ts gene are protected from both H-ras and BPV-1 transformation.

Human herpesvirus 6A (HHV-6A) strain U1102 was previously shown to contain a 1473 bp transformation suppressor gene (ts) (Araujo et al., 1995). Ts inhibited transformation of NIH3T3 cells by H-ras and transcription of the H-ras and human immunodeficiency type 1 (HIV-1) promoters in transient transfection experiments. In the current study, stable NIH3T3 cell lines expressing ts protein were established by transfection with pRc-ts containing the ts gene under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) and a neomycin selectable marker. Selected cell lines contained approximately one to two copies per cell of intact ts sequences, expressed ts protein and grew at approximately the same rate as parental NIH3T3 cells. These cell lines were protected from H-ras transformation while parental and NIH3T3 cells containing the ts gene cloned in the antisense orientation were not. Expression of the chloramphenicol acetyl transferase (CAT) gene under the control of the EJ-H-ras promoter was also suppressed in the ts cell lines but not when the CAT gene was under the control of the murine osteosarcoma virus LTR or human cytomegalovirus immediate early promoter. When NIH3T3 cell lines expressing ts protein were established by infection with the retrovirus, LNCts, the cells expressed ts protein and were protected from H-ras transformation. Furthermore, bovine papillomavirus type 1 (BPV-1) transformation was also suppressed in cells co-transfected with BPV-1 plus ts and in ts expressing cell lines transfected with BPV-1. The BPV-1 p89 and p2443 promoters were down-regulated in 3T3-ts lines. Because the human papillomavirus type 16 (HPV-16) p97 promoter has similarity to the BPV-1 p89 promoter, the ability of ts to suppress p97 was also tested. Like the H-ras and BPV-1 promoters, HPV-16 p97 was down-regulated in 3T3-ts lines. The data indicate the utility of ts against H-ras, BPV-1 and HPV-16 promoters and their respective oncogenes.

A transforming fragment within the direct repeat region of human herpesvirus type 6 that transactivates HIV-1.

HHV-6 infection has been associated with several malignancies including non-Hodgkin's lymphoma and Hodgkin's disease by the presence of high antibody titer and/or the presence of HHV-6 DNA. To understand their oncogenic potential, SalI restriction fragments from HHV-6 strain U1102 were transfected into NIH3T3 cells to assess transforming ability. A 3.9-kbp SalI-L DNA fragment spanning the junction of the direct repeat left (DRL) and unique long segment (UL) regions of HHV-6 induced foci of morphologically altered cells. The SalI-L transformed NIH3T3 focal lines induced tumors in nude mice within 2 weeks. The retention of HHV-6 specific DNA observed in SalI-L transformed cells and their tumor-derived lines suggest a possible maintenance function. Since both HHV-6 infection as well as transforming fragments from other DNA viruses have been shown to transactivate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR), SalI-L was examined for transactivation activity. SalI-L up-regulated HIV-1 LTR CAT 10-15 fold in both monkey CV-1 and human T Jurkat cells. The further study of the SalI-L transforming fragment exhibiting transactivation of HIV-1 LTR will elucidate whether these two activities are encoded by a single gene and will aid in the understanding of the interaction between HHV-6 and HIV-1 as it relates to progression of AIDS and/or AIDS-related malignancies.

Human herpesvirus 6 (HHV-6) ORF-1 transactivating gene exhibits malignant transforming activity and its protein binds to p53.

The 357 amino acid open reading frame 1 (ORF-1), also designated DR7, within the SalI-L fragment of human herpesvirus 6 (HHV-6) exhibited transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter and increased HIV-1 replication (Kashanchi et al., Virology, 201, 95-106, 1994). In the current study, the SalI-L transforming region was localized to the SalI-L-SH subfragment. Several ORFs identified in SalI-L-SH by sequence analysis were cloned into a selectable mammalian expression vector, pBK-CMV. Only pBK/ORF1 transformed NIH3T3 cells. Furthermore, cells expressing ORF-1 protein produced fibrosarcomas when injected into nude mice, whereas control cells, expressing either no ORF-1 protein or C-terminal truncated (after residue 172) ORF-1 protein, were not tumorigenic. Western blot analysis of proteins extracted from the tumors revealed ORF-1 protein. Additional studies indicated that ORF-1 was expressed in HHV-6-infected human T-cells by 18 h. Co-immunoprecipitation experiments showed that ORF-1 protein bound to tumor suppressor protein p53, and the ORF-1 binding domain on p53 was located between residues 28 and 187 of p53, overlapping with the specific DNA binding domain. Functional studies showed that p53-activated transcription was inhibited in ORF-1, but not in truncated ORF-1, expressing cells. Importantly, the truncated ORF-1 mutant also failed to cause transformation. Analysis of several human tumors by PCR revealed ORF-1 DNA sequences in some angioimmunoblastic lymphadenopathies, Hodgkin's and non-Hodgkin's lymphomas and glioblastomas. The detection of ORF-1 sequences in human tumors, while not proof per se, is a prerequisite for establishing its role in tumor development. Taken together, the results demonstrate that ORF-1 is an HHV-6 oncogene that binds to and affects p53. The identification of both transforming and transactivating activities within ORF-1 is a characteristic of other viral oncogenes and is the first reported for HHV-6.

HPLC purification of polymerase chain reaction products for direct sequencing.

Same day PCR amplification and sequencing is desired in situations where one needs to sequence a number of PCR products. The rapid, high-yield purification of PCR products via the use of high performance, anion-exchange chromatography yields sequencing results comparable to those obtained from techniques requiring subcloning of the PCR product. This can be achieved by standard dideoxynucleotide sequencing technology without the need to prepare prelabeled primers and additional internal primers or to gel purify the PCR product. In addition, this chromatographic technique offers the potential of isolating several PCR products from the same amplification mixture.

Transformation of Syrian hamster embryo cells by sodium bisulfite.

Exposure at neutral pH of Syrian hamster embryo cells (HEC) to non-lethal, non-mutagenic doses of bisulfite, the physiological form of SO2, causes a dose-dependent increase in transformation. When bisulfite is used in combination with UV irradiation, the induction of transformation is not synergistic. These results suggest that bisulfite transformation of HEC may not occur by a mechanism involving mutation.

Expression of initiated and promoted stages of irradiation carcinogenesis in vitro.

Evidence obtained utilizing X-irradiation and ultraviolet light irradiation with 12-O-tetradecanol-phorbol-13-acetate (TPA) for the induction of transformation of Syrian hamster embryo cells (HEC) suggests that there is more than 1 type of initiation of carcinogenesis. Initiated HEC are phenotypically transformed in the presence of TPA (15--20-fold enhancement) but are not necessarily committed to be transformed because some colonies reverted to normal morphology when TPA was removed. Both initiated and promoted HEC are also sensitive to hamster lymphotoxin. The frequency of transformation was reduced by at least 70%. The ability to recognize discrete morphologically altered colonies after TPA addition in vitro indicates that promotion is not merely escape from growth control.

9-nitrocamptothecin selectively inhibits human immunodeficiency virus type 1 replication in freshly infected parental but not 9-nitrocamptothecin-resistant U937 monocytoid cells.

We have previously reported that 9-nitrocamptothecin (9NC) inhibited human immunodeficiency type 1 (HIV-1) replication in latently HIV-1-infected T lymphocytic ACH-2 cells stimulated with the cytokine tumor necrosis factor alpha (TNF-alpha) (Moulton et al., AIDS Res Hum Retroviruses 1998;14:39). 9NC induced an accelerated apoptosis in HIV-1-infected, but not uninfected, lymphocytic cells. The present study demonstrates that 9NC selectively inhibits release of HIV-1 from freshly infected monocytoid U937 cells in a dose-response manner. Significant inhibition was achieved with concentrations of 9NC that were not toxic. In contrast, HIV-1 replication in 9NC-resistant monocytoid cells, derived from U937, was not inhibited by similar doses of 9NC. Importantly, sensitivity of HIV-1 replication to 9NC correlated with the effect of 9NC on topoisomerase I (topo I) activity. In a 9NC-sensitive subline, 9NC induced posttranslational activation of the nuclear transcription factor kappaB (NF-kappaB) after the drug treatment. This activation was neither related to selective 9NC suppression of HIV-1 replication, nor was it sufficient for the 9NC-induced toxicity in the drug-sensitive monocytoid cells. Taken together, the selective inhibition of HIV-1 replication in both lymphoid and monocytoid cells lends further credence to the potential development of 9NC as an alternative drug for treating HIV-1 infection.

Characterization of the human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) oncogene, kaposin (ORF K12).

BACKGROUND: Human herpesvirus 8 (HHV-8) has been implicated in the etiology of Kaposi's sarcoma (KS), a highly angiogenic tumor of complex histology, and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). A number of HHV-8 encoded genes have been proposed to be involved in the pathogenesis of KS and PEL and a few have been shown to be oncogenic in heterologous systems (Reyes GR, LaFemina R, Hayward SD, Hayward GS. Morphological transformation by DNA fragments of human herpesviruses: evidence for two distinct transforming regions in herpes simplex virus types 1 and 2 and lack of correlation with biochemical transfer of the thymidine kinase gene. Cold Spring Harbor Symp Quant Biol 1980;44:629-641; Moore PS, Boshoff C, Weiss RA, Chang Y. Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV. Science 1996;274:1739-1744; Cheng EH, Nicholas J, Bellows DS, Hayward GS, Guo HG, Reitz MS, Hardwick JM. A Bcl-2 homolog encoded by Kaposi sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Bax or Bak. Proc Natl Acad Sci USA 1997;94:690-694; Li M, Lee H, Yoon DW, Albrecht JC, Fleckenstein B, Neipel F, Jung JU. Kaposi's sarcoma-associated herpesvirus encodes a functional cyclin. J Virol 1997;71:1984-1991; Neipel F, Albrecht J-C, Fleckenstein B. Cell-homologous genes In the Kaposi's sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity? J Virol 1997;71:4187-4192; Nicholas J, Ruvolo VR, Burns WH, Sandford G, Wan X, Ciufo D, Hendrickson SB, Guo HG, Hayward GS, Reitz MS. Kaposi's sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6. Nat Med 1997;3:287-292; Nicholas J, Zong J, Alcendor DJ, Ciufu DM, Poole LJ, Sarisky RT, Chiuo C, Zhang X, Wan X, Guo H, Reitz MS, Hayward GS. Novel organizational features, captured cellular genes, and strain variability within the genome of KSHV/HHV-8. JNCI Monographs 1998;23:79-88; Muralidhar S, Pumfery AM, Hassani M, Sadaie MR, Azumi N, Kishishita M, Brady JN, Doniger J, Medveczky P, Rosenthal LJ. Identification of kaposin (ORF K12) as a human herpesvirus 8 (Kaposi's sarcoma associated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The kaposin gene (ORF K12) encoded by the abundant latency-associated HHV-8 transcript, T0.7, has been previously shown to induce tumorigenic transformation of Rat-3 cells (Muralidhar S, Pumfery AM, Hassani M, Sadaie MR, Azumi N, Kishishita M, Brady JN, Doniger J, Medveczky P, Rosenthal LJ. Identification of kaposin (ORF K12) as a human herpesvirus 8 (Kaposi's sarcoma associated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The current study is a further characterization of kaposin protein. OBJECTIVES: Characterization of kaposin expression in transformed and tumor-derived Rat-3 cells as well as PEL cell lines, BCBL-1, BC-3 and KS-1 and analysis of mechanism(s) of transformation. DESIGN: The presence of kaposin DNA in transformed cells was determined by fluorescent in situ hybridization (FISH). Expression of kaposin protein was analyzed by Western blot analysis and indirect immunofluorescence assay (IFA). (ABSTRACT TRUNCATED)