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Z Z Su

Publications and source records attributed to Z Z Su.

At least 37 records · Page 2Linked to original sources
PubMed1994

Induction of transformation progression in type 5 adenovirus-transformed rat embryo cells by a cloned protein kinase C beta 1 gene and reversal of progression by 5-azacytidine.

Protein kinase C (PKC) is a key component in signal transduction in eucaryotic cells and when specific PKC isoforms are over-expressed in immortal mammalian cells they can induce transformation-associated properties. In the present study we demonstrate that a cloned PKC beta 1 gene can induce an enhanced expression of the transformed phenotype in type 5 adenovirus (Ad5)-transformed rat embryo (RE) cells (clone E11), a process termed transformation progression. E11 cells expressing the PKC beta 1 gene, clone B1/PKC, produce PKC beta 1 mRNA and display enhanced PKC enzymatic activity and binding of [3H]-phorbol-12,13-dibutyrate (PDBu) to cell surface phorbol ester receptors. B1/PKC cells grow with increased efficiency in agar in comparison with parental E11 cells and anchorage-independence is further enhanced in both cell types by addition of the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A single-exposure of B1/PKC cells to 5-azacytidine (AZA), followed by growth in the absence of this demethylating agent, results in B1/PKC-AZA clones which display a stable reversion of the progression phenotype to that of the unprogressed parental E11 clone. Loss of the progression phenotype corresponds with a reduction in PKC beta 1-induced biochemical and cellular changes. In contrast, progression-suppression does not involve an alteration in expression of the Ad5 transforming genes, E1A and E1B, or the endogenous PKC epsilon gene. TPA cannot induce the progression phenotype in B1/PKC-AZA cells, but it can reversibly induce an increase in the transcriptional rate and steady-state mRNA levels of PKC beta 1 and c-jun and it increases AP-1 DNA-binding. These results indicate that the PKC beta 1 gene can serve as a transformation progression-inducing gene in rat embryo cells previously transformed by Ad5 and progression may be mediated by the inactivation by methylation of an AZA-sensitive 'progression suppressor gene(s)'. The suppression process in B1/PKC cells is independent of expression of the Ad5-transforming genes but correlates directly with the reduced expression of the transfected PKC beta 1 gene in AZA-treated B1/PKC cells.

Animals↗
PubMed1994

Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53.

The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed in Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.

Cell Differentiation↗
PubMed1993

Wild-type adenovirus type 5 transforming genes function as transdominant suppressors of oncogenesis in mutant adenovirus type 5 transformed rat embryo fibroblast cells.

Transformation of cloned rat embryo fibroblast (CREF) cells with the host-range adenovirus type 5 (Ad5) mutant, H5hr1, results in transformants with a fibroblastic morphology which displays a cold-sensitive transformation phenotype and oncogenic potential in both nude mice and syngeneic rats. In contrast, wild-type (wt) Ad5 transformed CREF cells are epithelioid in morphology, temperature independent for transformation, and nontumorigenic. The present studies were conducted to analyze the contribution of the mutated E1A and E1B regions of H5hr1 in regulating the biological properties of H5hr1-transformed CREF cells. CREF cells were constructed which contain the mutated E1A and E1B transforming regions of H5hr1 and either a wt Ad5 E1A gene, a wt Ad5 E1B gene, or both a wt Ad5 E1A and a wt E1B gene. A wt Ad5 E1A gene was sufficient in reversing the cold-sensitive transformation phenotype. By using a wt Ad5 E1A gene under the transcriptional control of a dexamethasone-inducible mouse mammary tumor virus promoter, a direct suppressive effect of wt Ad5 E1A on colony formation in monolayer culture and agar growth of H5hr1-transformed cells was demonstrated. Expression of a wt Ad5 E1A, a wt Ad5 E1B, or both wt transforming genes in H5hr1-transformed CREF cells also suppressed oncogenicity. The ability or inability to form tumors in animals was found not to correlate with sensitivity to natural killer cell-mediated lysis. These results indicate that both the wt Ad5 E1A and wt Ad5 E1B genes can function as dominant suppressors of the oncogenic process when coexpressed in H5hr1-transformed CREF cells. This effect does not require large quantities of wt Ad5 E1A or E1B transforming proteins, nor is it directly related to the acquisition of a natural killer cell cytolysis-susceptible phenotype.

Adenovirus E1A Proteins↗
PubMed1993

Genetic analysis of carcinogen enhancement of type 5 adenovirus transformation of cloned Fischer rat embryo fibroblast cells.

Pretreatment of CREF cells with methyl methanesulfonate (MMS) before infection with the host-range cold-sensitive type 5 adenovirus (Ad5) mutant H5hr1 results in a dose-dependent carcinogen enhancement of viral transformation (CET). The properties of CET observed with H5hr1, which include both an MMS dose-dependent enhancement in the number of transformed foci and an increase in transformation frequency after correction for cell toxicity, are not observed in carcinogen-pretreated wild-type (wt) Ad5 (H5wt)-infected CREF cells. This study was conducted to determine the role of the viral E1A and E1B transforming genes of H5hr1 in mediating the unique CET phenotype of H5hr1. Coinfection of MMS-pretreated CREF cells with H5wt or H5sub309 (which displays a wt Ad5 phenotype) and H5hr1 resulted in a suppression of the unique CET phenotype that was directly related to the multiplicity of infection with wt Ad5. Suppression of the unique H5hr1 CET phenotype was also apparent in MMS-pretreated CREF cells coinfected with H5hr1 and an Ad5 mutant expressing either a wt 13S E1A-encoded 289 amino-acid (aa) protein and an intact wt E1B gene or a wt 13S E1A-encoded 289-aa protein and a 22S E1B-encoded 495-aa protein. In contrast, the unique H5hr1 CET phenotype was not suppressed in MMS-pretreated CREF cells coinfected with H5hr1 and Ad5 or Ad2 mutants expressing either a wt 12S E1A-encoded 243-aa protein and both wt E1B gene products or an intact wt E1A gene and a wt E1B 13S-encoded 175-aa protein. That genetic changes in both the E1A and E1B viral regions of H5hr1 were required to induce the unique CET phenotype was also indicated by the inability of a recombinant Ad5 containing the 0-4.5 map-unit region of H5hr1 and the 4.5-100 map-unit region of H5sub309 to display the H5hr1 unique CET phenotype. Direct confirmation of the requirement for both gene regions of H5hr1 to mediate its unique CET was obtained by generating CREF cells stably expressing a wt Ad5 E1A 13S-encoded 289-aa protein and a wt E1B 22S-encoded 495-aa protein. In these CREF transformants (which displayed a CREF-like morphology), transformation by H5hr1 was not reduced, but the unique CET phenotype after MMS pretreatment was eliminated. These results suggest that alterations in both the 13S-encoded E1A and 22S-encoded E1B gene products of H5hr1 contribute to its unique CET.

Adenoviridae↗
PubMed1993

[Clinical and experimental study on effects of man-shen-ling oral liquid in the treatment of 100 cases of chronic nephritis].

A new Chinese herbal preparation, Man-Shen-Ling (MSL, consisted of medicinal herbs such as Astragalus and Rehmannia) in treating 100 cases of chronic nephritis. The effective rate was 91% in comparing to 66.7% in the control group, P < 0.001. It was markedly effective for proteinuria, hematuria, improvement and recovery of renal functions, edema, anemia, anorexia etc in comparing with the control group. It showed no adverse effects on functions of liver, kidney, heart and GI tract. Animal model of chronic nephritis was established and the effects of MSL were observed. The laboratory findings and histopathological investigation on kidney revealed and confirmed that MSL has therapeutic effects on chronic nephritis. Pharmacodynamically, MSL exhibited effects of anti-allergy, its immuno-suppressive effect corresponded to that of cyclophosphamide, with diuretic, hypotensive, proteinuria eliminating, anti-inflammatory, anti-coagulatory, renal blood flow and glomerular filtration enhancing, the excretion of urea-nitrogen, potassium and sodium promoting function; in addition, it also could promote and modulate the immunity. Acute and chronic toxicity tests on animal models neither showed toxic, mutagenic, teratogenic nor carcinogenic effects. It is a new preparation of Chinese medicinal herbs in treating chronic nephritis, it is safe and effective.

Adolescent↗
PubMed1993

Defining the critical gene expression changes associated with expression and suppression of the tumorigenic and metastatic phenotype in Ha-ras-transformed cloned rat embryo fibroblast cells.

Carcinogenesis requires a complex series of genetic changes often involving multiple oncogenes and the inactivation of multiple tumor-suppressor genes. We presently examined the effect of the Krev-1 tumor-suppressor gene on the tumorigenic and metastatic potential of Ha-ras-transformed cloned rat embryo fibroblast (CREF) cells. Ha-ras-transformed CREF cells are morphologically transformed and anchorage independent; produce reduced levels of nm23-H1 (a putative metastasis-suppressor gene product) and TIMP-1 (tissue inhibitor of metalloproteinase 1) transcripts and mRNA compared with CREF cells; produce increased levels of cripto, 94-kDa gelatinase/type IV collagenase (94-kDa GEL), osteopontin (OPN) and transin/stromelysin transcripts and mRNA compared with CREF cells; and are tumorigenic and metastatic in both nude mice and syngeneic rats. Ha-ras-transformed CREF cells coexpressing the Krev-1 gene display a reversion in cellular phenotype and gene expression to that of untransformed CREF cells. However, Ha-ras/Krev-1-coexpressing CREF cells retain, albeit with extended latency periods, both tumorigenic and metastatic potential that is not related directly to the final level of Ha-ras or Krev-1 mRNA or the Ha-ras p21 transforming protein. Development of metastatic potential is, however, directly correlated with a reduction in nm23-H1 and TIMP-1 transcription and mRNA levels and an enhanced expression of cripto, 94-kDa GEL, osteopontin and transin. In contrast, expression of additional tumor-suppressor genes, such as the RB gene and p53, or genes associated with tumorigenesis in other model systems, such as major excreted glycoprotein (MEP), 72-kDa gelatinase/type IV collagenase (72-kDa GEL), fibronectin (FIB), tenascin and intracellular adhesion molecule 1 (ICAM-1) is not altered in a consistent manner during in vitro transformation suppression or escape from tumorigenic and metastatic suppression. These results indicate that Krev-1 suppression of the Ha-ras-transformed/oncogenic phenotype is associated with a distinct program of gene expression changes manifested by altered rates of transcription and steady-state mRNA levels of specific oncogenic-suppressing and oncogenic-inducing genes. These data support a model of Ha-ras-induced metastasis in CREF cells that involves a direct modulation in the expression/suppression of specific combinations of oncogenic-suppressor genes and metastasis-promoting genes that are regulated coordinately in the process of tumor progression.

Animals↗
PubMed1992

Growth inhibition and modulation of antigenic phenotype in human melanoma and glioblastoma multiforme cells by caffeic acid phenethyl ester (CAPE)

The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), has been shown to display increased toxicity toward various oncogene-transformed cell lines in comparison with their untransformed counterparts (Su et al., 4: 231-242, 1991). This observation provides support for the concept that it is the transformed phenotype which is specifically sensitive to CAPE. In the present study, we have determined the effect of CAPE on the growth and antigenic phenotype of a human melanoma cell line, HO-1, and a human glioblastoma multiforme cell line, GBM-18. For comparison, we have also tested the effects of mezerein (MEZ), mycophenolic acid (MPA) and retinoic acid (RA), which can differentially modulate growth, differentiation and the antigenic phenotype in these human tumor cell lines. Growth of both cell lines was suppressed by CAPE in a dose-dependent fashion, with HO-1 cells being more sensitive than GBM-18 cells. The antiproliferative effect of CAPE was enhanced in both cell types if CAPE and MEZ were used in combination. Growth suppression was associated with morphological changes in H0-1 cells, suggesting induction of a more differentiated phenotype. CAPE also differentially modulated the expression of several antigens on the surface of the two tumor cell lines. These results suggest a potential role for CAPE as an antitumor agent, an antigenic modulating agent and possibly a differentiation inducing agent.

Antigens, Neoplasm↗
PubMed1992

Alterations in cellular phenotype induced by the type 5 adenovirus E1A and the beta 1 protein kinase C genes in cloned rat embryo fibroblast cells.

The E1A gene of adenovirus type 5 (Ad5) induces morphological transformation and anchorage-independent growth in cloned rat embryo fibroblast (CREF) cells. In contrast, CREF cells transfected with a beta 1 protein kinase C (PKC) gene and expressing low-levels of beta 1 PKC display a CREF-like morphology and do not form colonies when grown in agar. The combination of Ad5 E1A and low-level beta 1 PKC expression in the same CREF cell line results in an enhanced ability to grow when suspended in agar. In Ad5 E1A and Ad5 E1A + low-level beta 1 PKC expressing CREF clones, the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further enhances anchorage-independence. In contrast, TPA does not induce CREF cells or transfected CREF cells expressing low-levels of beta 1 PKC to grow in agar. Low-level beta 1 PKC expression in transfected CREF cells is associated with a modest 1.2 to 1.6-fold increase in binding of [3H]-phorbol-12,13-dibutyrate (PDBu) and only a 2.3-fold increase in PKC enzymatic activity. In contrast, specific beta 1 PKC-retroviral vector transformed CREF clones (CREF-RV-PKC) display higher levels of PKC mRNA, PDBu binding and PKC enzymatic activity. A majority of CREF-RV-PKC clones exhibit a transformed morphology and grow more rapidly in monolayer culture, form macroscopic colonies in agar in the absence of TPA and in many independent clones TPA further enhances anchorage-independent growth. This effect is not directly related to the level of enhanced [3H]-PDBu binding. The present study indicates that the effect of beta 1 PKC on cellular phenotype in immortal rat embryo cells is complex and is affected by its mode of insertion into CREF cells, i.e. transfection versus retroviral insertion. In addition, the combination of a transfected Ad5 E1A and a beta 1 PKC gene in the same CREF clone results in an enhanced expression of the transformed phenotype in both the absence and presence of TPA.

Adenoviridae↗
PubMed1991

Suppression of adenovirus type 5 E1A-mediated transformation and expression of the transformed phenotype by caffeic acid phenethyl ester (CAPE).

Viral transformation and DNA-transfection assays were employed to investigate the differential toxic effect of caffeic acid phenethyl ester (CAPE), an extract of the honeybee hive product propolis, on adenovirus type 5 (Ad5)-transformed cloned rat embryo fibroblast (CREF) cells. CAPE inhibited, in a dose-dependent manner, both de novo and carcinogen-enhanced transformation of CREF cells by H5hr1, the cold-sensitive (cs) host-range mutant of Ad5. CAPE had a selective inhibitory effect on Ad5-induced transformation when a wild-type (wt) Ad5 E1A gene or a cs Ad5 E1A gene (at 37 degrees C, but not at 32 degrees C) was cotransfected into CREF cells with a dominant-acting bacterial hygromycin-resistance gene. A requirement for the expression of Ad5 E1A-encoded mRNAs and transforming proteins and sensitivity to CAPE was demonstrated using CREF cells stably transformed by a cs Ad5 E1A gene and an Ad5 E1A gene under the transcriptional control of a mouse mammary tumor virus promoter. To distinguish between the effects of the two Ad5 E1A-encoded proteins of 289 amino acids (aa) and 243 aa, CREF cells were stably transformed with cDNAs encoding either the 13S or the 12S E1A mRNA. CREF cells expressing the 13S E1A-encoded 289-aa protein were more sensitive to the growth-suppressing effect of CAPE than cells producing only the 12S E1A-encoded 243-aa protein. However, the growth-suppressing and toxic effects of CAPE were greatest in cells expressing both E1A-encoded transforming proteins. Analysis of the effect of CAPE on E1A and beta-actin gene expression in wt and cs E1A and H5hr1-transformed CREF cells indicated that low levels of CAPE, which were growth suppressive, did not selectively suppress E1A expression. These results demonstrated that cellular changes induced in CREF cells by the 13S E1A-encoded 289-aa protein of Ad5, when expressed alone or in combination with the 12S E1A-encoded 243-aa protein, rendered transformed cells sensitive to the growth-suppressing and toxic effects of CAPE.

Actins↗
PubMed1991

Low-level beta 1 protein kinase C expression in cloned rat embryo fibroblast cells enhances transformation induced by the adenovirus type 5 E1A gene.

Expression of the E1A gene of adenovirus type 5 (Ad5) in a cloned rat embryo fibroblast (CREF) cell line results in morphological transformation. The efficiency of E1A-mediated transformation of CREF cells is increased if a wild-type Ad5 E1A gene is cotransfected with a rat beta 1 protein kinase C (beta 1 PKC) gene. A direct demonstration of complementation between a functional-transforming Ad5 E1A gene and beta 1 PK in inducing transformation was demonstrated using Ad5 E1A cold-sensitive mutant (E1Acs) genes. The E1Acs gene enhanced transformation only at the transformation-permissive temperature of 37 degrees C and not at the nonpermissive transforming temperature of 32 degrees C. CREF cells constitutively expressing low levels of beta 1 PKC mRNA were transformed at a higher frequency than parental CREF cells after transfection with an Ad5 E1A gene or infection with wild-type Ad5 or the Ad5 host-range cold-sensitive mutant H5hr1. There was no enhancement of transformation in low-level beta 1 PKC-expressing CREF cells when cultures were grown continuously in the presence of the PKC-inhibitor 1-(5-isoquinolynsulfonyl)-2-methylpiperazine dihydrochloride. Transfected CREF cells expressing low levels of beta 1 PKC mRNA displayed CREF-like morphology and did not form colonies when grown in agar. In contrast, retroviral vector-transformed CREF cells expressing high levels of beta 1 PKC mRNA and beta 1 PKC enzyme activity were morphologically transformed and grew efficiently in agar. These findings indicate that the beta 1 PKC gene, when expressed at low levels, can cooperate with the Ad5 E1A gene in the initiation of viral oncogene-mediated transformation.

Adenovirus Early Proteins↗
PubMed1991

Analysis of viral and cellular gene expression during progression and suppression of the transformed phenotype in type 5 adenovirus-transformed rat embryo cells.

Transformation of secondary Sprague-Dawley rat embryo (RE) cells with type 5 adenovirus (Ad5) results in morphologically transformed cells which can undergo a series of sequential changes resulting in enhanced expression of the transformed phenotype, a process termed progression. Selection for a progressed phenotype often occurs after growth in agar or tumor formation in nude mice, and this process is reversible following treatment of cells with 5-azacytidine. In the present study we have analyzed a series of clonal populations of Ad5-transformed RE cells representing different stages in a defined progression lineage. Progression was not associated with alterations in the steady-state levels of mRNA produced by the viral transforming genes, E1A and E1B, or the cellular gene, c-myc. In addition, the tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which induces expression of a progressed phenotype in Ad5-transformed RE cells, did not significantly alter the RNA transcription rates of the Ad5 E1A or E1B genes, the TPA-inducible gene TPA-S1 or the TPA-responsive genes Pro1 or protein kinase C. TPA did, however, increase by 1 h the steady-state level of c-fos mRNA, but this effect was similar in both progressed and unprogressed cells. Progression also did not involve a change in the RNA transcription rate of a number of cellular and viral genes, including actin, c-Ha-ras, c-myc, v-fos, erbB, TGF-alpha, TGF-beta, Pro-2, transin, TPA-R1, v-myb and c-mos, or other adenovirus genes in addition to E1A and E1B, including E2A and E4. Immunoblotting analysis using E1B polyclonal antiserum further indicated that progression was not associated with changes in the levels of an Mr 21,000 polypeptide encoded by E1B. Similarly, immunoprecipitation analysis with an Ad2 E1A monoclonal antibody indicated similar levels of the Mr 55,000 and 48,000 E1A polypeptides, as well as coprecipitated proteins of Mr 300,000, 107,000 and 105,000 [which is the retinoblastoma (Rb) protein], in E11 and E11-NMT cells. Immunoprecipitation of cell lysates with a monoclonal antibody specific for the Mr 105,000 Rb protein further demonstrated that progression also was not associated with a change in the level or state of phosphorylation of the Rb protein. However, transfection of a human Rb gene (also containing a neomycin resistance gene) into Ad5-transformed RE cells was more inhibitory, with respect to formation of G418-resistant colonies, in unprogressed than in progressed Ad5-transformed RE cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗
PubMed1990

Enhancement of adenovirus transformation of cloned rat embryo fibroblast cells by gamma irradiation.

We have analyzed the effect of gamma irradiation on the induction of morphological transformation of cloned rat embryo fibroblast (CREF) cells by the host-range cold-sensitive type 5 adenovirus mutant, H5hr1. Treatment of CREF cells with 1-6 Gy of gamma irradiation immediately prior to viral infection resulted in dose-dependent decrease in cell survival and concomitant increase in viral transformation frequency. Exposure of CREF cells to 1-6 Gy of gamma radiation alone resulted in a similar dose-dependent inhibition in cell survival but without any subsequent morphological transformation. The effect of gamma irradiation on viral transformation was greatest when cells were irradiated directly before viral infection. The reduction in the enhancement of transformation was both dose and time dependent. The ability of gamma irradiation to enhance viral transformation was substantially reduced if CREF cells were treated with inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Employing a single-cell colony transfer assay and in situ hybridization with a 32P-labeled Ad5 DNA probe, we found that gamma irradiation of CREF cells prior to infection with H5hr1 resulted, 10 and 17 d after infection and replating, in an increase in the percentage of surviving CREF colonies that contain Ad5 DNA. Analysis of viral DNA integration by DNA-filter hybridization (Southern blot analysis) in H5hr1-transformed CREF clones isolated from untreated and gamma-irradiated cultures indicates that gamma irradiation caused increases in both the number of copies of Ad5 E1A DNA sequences integrated into cellular DNA and the number of unique Ad5 E1A DNA integration sites in transformed cells. These results indicate that gamma irradiation enhancement of adenovirus transformation was a consequence of radiation-induced cellular factors with finite life spans that are mediators of enhanced viral transformation. Potentially important components of the radiation enhancement process appear to involve an alteration in both the retention of free Ad5 DNA in surviving cells and an alteration in the profile of viral-DNA integration in gamma-irradiated cells.

Adenoviridae↗
PubMed1990

Enhancement of viral and DNA mediated transformation of cloned rat embryo fibroblast cells by 3-aminobenzamide.

We have analyzed the effect of the poly(ADP-ribose) synthesis inhibitor 3 aminobenzamide (3AB) on de novo and methyl methanesulfonate (MMS) and gamma irradiation enhancement of viral transformation of a cloned rat embryo fibroblast cell line, CREF, by a cold-sensitive host-range mutant of type 5 adenovirus, H5hr1. Additionally, we have evaluated the effect of 3AB on the transformation of CREF cells following transfection with a gene conferring resistance to hygromycin (hygr) or the neomycin analogue G418 (neor) in combination with a cloned type 5 adenovirus E1A transforming gene (Ad5 E1A) or the Ha-ras (T24) oncogene. 3AB induced a dose- and time-dependent increase in the level of de novo MMS-enhanced and gamma irradiation-enhanced transformation of CREF cells by H5hr1, whereas it did not induce morphological transformation in uninfected control, MMS-pretreated, or gamma irradiation-pretreated CREF cells. Temporal kinetic studies indicated that 3AB was most effective in enhancing de novo and MMS-enhanced and gamma irradiation-enhanced viral transformation when applied early after viral and carcinogen plus viral infection and when present for extended time periods (4-5 wk). 3AB also increased the frequency of resistant colonies following transfection with several cloned genes, including hygr, neor, and protein kinase C (which also contained a neor gene), and the frequency of morphological transformation of CREF cells following cotransfection with a hygr gene and an Ad5 E1A or an activated Ha-ras (T24) gene. In contrast, 3AB exerted the opposite effect, i.e., an inhibitory effect, when applied to NIH 3T3 cells transfected with a hygr or neor gene, alone or in combination with a T24 gene. The ability of 3AB to enhance the frequency of transformation of CREF cells was not associated with a selective effect on the growth of H5hr1-transformed CREF cells in monolayer or agar culture. Similarly, 3AB did not alter the percentage of MMS- or gamma irradiated-pretreated H5hr1-infected cells retaining free Ad5 DNA or the random pattern or quantity of viral DNA integration in control or carcinogen-treated H5hr1-transformed cells. These results suggest that cellular processes regulated by the nuclear enzyme ADPRT, or additional processes modified by 3AB, may be important mediators of stable transformation induced by transfected DNA and both de novo and carcinogen-enhanced viral transformation of specific target cells.

Adenoviridae↗
PubMed1989

Early events in methyl methanesulfonate enhancement of adenovirus transformation of cloned rat embryo fibroblast cells.

Pretreatment of a cloned rat embryo fibroblast (CREF) cell line with methyl methanesulfonate (MMS) prior to infection with a specific host-range and cold-sensitive type 5 adenovirus mutant (H5hr1), results in a unique carcinogen enhancement of transformation (CET) phenotype (Carcinogenesis 8:967, 1987). By using low-density clonal assays and in situ hybridization techniques with 32P-labeled type 5 adenovirus (Ad5) probes, we demonstrated that 5-10 d following infection the proportion of CREF colonies containing H5hr1 DNA and RNA is increased two- to threefold as a result of pretreatment with MMS. Twenty-five days following infection of CREF cells, Ad5 DNA assays showed that both solvent and MMS-pretreated CREF colonies no longer contained detectable levels of viral DNA or RNA. Analysis of free viral DNA by the Hirt procedure suggested that more free viral DNA persisted in MMS-pretreated H5hr1-infected CREF cells than in solvent-pretreated H5hr1-infected CREF cells. The relative amount of free viral DNA in both types of cultures was directly related to the multiplicity of H5hr1 infection and decreased with time following infection. As observed using in situ hybridization techniques, by 25 d after infection no free viral DNA was detected in either MMS- or solvent-pretreated H5hr1-infected CREF cells. By using a protein synthesis inhibitor (cycloheximide) and an RNA transcription inhibitor (actinomycin D), it was further demonstrated that the ability of MMS to induce a unique CET in CREF cells following infection with H5hr1 was dependent on the synthesis of new protein and RNA. In contrast, inhibition of protein and RNA synthesis did not alter the de novo rate of H5hr1 transformation of CREF cells. Temporal kinetic studies indicated that the ability of MMS to enhance H5hr1 transformation of CREF cells and to increase the percentage of CREF colonies containing Ad5 genetic information is regulated in a strict temporal manner. The results of the present investigation suggest that the ability of MMS to enhance H5hr1 transformation of CREF cells is dependent on the induction of new protein(s) in CREF cells, and enhancement is associated with an increase in the proportion of cells in the infected CREF cell population that initially contain Ad5 DNA/RNA.

Adenoviruses, Human↗
PubMed1988

Positive selection of human cells lacking several transformation parameters from an SV40-transformed culture by means of parvovirus H-1.

The simian virus 40 (SV40)-transformed, newborn human kidney cell line NB-F was found to be heterogeneous with respect to its sensitivity to parvovirus H-1. The majority of the cells sustain a productive H-1 infection which eventually causes their lysis. Yet, a small fraction of the cells appears to be much less susceptible to H-1. Such a resistance to H-1 infection is a stable, transmissible property of this subpopulation of cells which was denoted NB-FR. The heterogeneity of NB-F cells is also apparent from the distribution of their karyotypes, which is bimodal and peaks at 114 and 46 chromosomes/cell. In contrast, the great majority of NB-FR cells contain 41-50 chromosomes. H-1-resistant and -sensitive cells appear to be related in several respects: they both contain morphologically human chromosomes as well as multiple SV40 DNA inserts, and could not be distinguished by isoenzyme typing. It was investigated whether the degree of sensitivity to H-1 infection correlated with other phenotypic properties of the human cell derivatives. NB-F cultures exhibit a series of transformation parameters, such as SV40 T-antigen expression, poor contact inhibition, clonogenicity in semi-solid medium and high lectin agglutinability, which are all much reduced or even undetectable in NB-FR cells. These observations suggest that cell susceptibility to H-1 segregates with marker(s) of in vitro malignant transformation. Moreover, the data indicate that parvoviruses can be used to preferentially remove transformants from a mixed culture of normal and transformed cells.

Antigens, Viral, Tumor↗
PubMed1988

Inhibitory effect of parvovirus H-1 on cultured human tumour cells or transformed cells.

The present study establishes the in vitro system for studying the oncosuppression activity of parvovirus and discovers that parvovirus H-1 can be grown cytolytically in various human cancer cell lines, which include 4 hepatoma cell lines (QGY-7703, SMMC-7721, Bel-7402, PLC/PRF/5), 3 gastric cancer cell lines (SGC-7901, MGC-80-3, MKN-28) and 1 naspharynx cancer cell line (CNE). The growth of two primary gastric cancer cell cultures from surgical cancer tissue were also inhibited by the infection of H-1. The sensitivity of cancer cells to H-1 may relate to their differentiation states. On the contrary, H-1 can neither be grown cytolytically in normal liver or stomach cells, nor inhibit their growth. Transformation of human skin fibroblasts with Simian Virus 40 activated their sensitivity to H-1. Our results thus indicate that the antineoplastic activity of H-1 in vivo involves at least its direct inhibiting or killing malignant cells.

Carcinoma, Hepatocellular↗
PubMed1988

Transformation of cultured human fetal gastric fibroblasts with physico-chemical agents.

Treatment of early passage human fetal gastric fibroblasts with ultraviolet (UV) light and the chemical carcinogen ethyl nitrosourea (ENU) in succession resulted in an immortally growing cell line, named GTS 8502. The cells of this line display typical transformation characteristics, such as irregularly shaped nuclei, heteroploidization of karyotype and frequent appearance of heteromorphic chromosomes, the enhanced volume ratio of nucleus to cytoplasm, multinucleoli, appearance of microvilli on the surface of the cells and agglutination reaction to lectin concanavalin A. The transformants have high growing and mitotic indices and the ability of focus-formation on monolayers and anchorage independent growth in soft agar medium. Moreover, these cells induced tumours in nude mice or in immunosuppressed new-born rats through heterotransplantation. The results of various methods including electromicroscopy and histochemical analyses indicate that GTS 8502 cells are of fibroblast origin. Our results thus indicate that synergism of two carcinogens may raise the transformation ratio of normal human cells, which is otherwise extremely low. The transformation of human cells may be utilized to detect environmental carcinogens.

Animals↗