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PubMedDate not supplied

Ad-fRNK and Ad-p53 cooperate to augment drug-induced death of a transformed cell line.

BACKGROUND: Transformed cells have abnormalities in the survival pathways contributing to drug resistance. These abnormalities include p53 mutations and increased focal adhesion kinase (FAK) expression. Because FAK regulates cell survival via p53-dependent and -independent pathways, it was hypothesized that combined therapy using wild-type p53 and an inhibitor of FAK (FRNK) would sensitize cells to anticancer drugs. MATERIALS AND METHODS: RPMI 2650 cells were infected with recombinant adenoviruses causing overexpression of p53 (Ad-p53) and FRNK (Ad-FRNK). Cell viability and apoptosis were measured using in vitro assays, whereas protein expression was determined by Western blotting. RESULTS: Co-infection of cells with Ad-p53 and Ad-FRNK induced more cellular apoptosis than transfection with either agent alone. Likewise, the co-transfection of cells with Ad-FRNK and Ad-p53 improved the cytotoxic response to four commonly used anticancer drugs relative to cells transfected with Ad-FRNK alone, Ad-p53 alone, or the equivalent amount of control adenovirus. This effect was associated with loss of endogenous FAK protein.

Adenoviridae↗
PubMed2004

Down-regulation of SHP1 and up-regulation of negative regulators of JAK/STAT signaling in HTLV-1 transformed cell lines and freshly transformed human peripheral blood CD4+ T-cells.

Adult T-cell leukemia (ATL) is an aggressive malignancy that is associated with human T-cell lymphotropic virus I (HTLV-I) infection. HTLV-I transformed T-cell lines and fresh ATL cells are characterized by constitutive activation of the interleukin-2 receptor (IL-2R) signaling pathway however, the mechanism(s) responsible for constitutive IL-2R activation are unknown. To further examine the cause of this signaling pathway deregulation, we measured mRNA and protein expression levels by real-time PCR and Western blots, respectively, of four negative regulators of the IL-2R signaling pathway including src homology 2 (SH2)-containing phosphatase (SHP1), cytokine-inducible (CIS) SH2-containing protein, suppressor of cytokine signaling-1 (SOCS1) and protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3) (PIAS3) in six HTLV-1 negative and seven HTLV-1 positive T-cell leukemia lines. The activation status of the JAK/STAT pathway was also examined. SHP1 mRNA and protein expression levels were selectively down regulated in all HTLV-1-infected transformed cell lines, while CIS, SOCS1, and PIAS3 protein expression were markedly but variably upregulated and the cells showed evidence of constitutive STAT3 activation. In acutely HTLV-1 infected primary CD4+ T-cells there was a gradual loss of SHP1 expression over 10 weeks in culture which correlated with progression from immortalization to transformation and loss of IL-2 dependence for growth. Two transformed cell lines that were established following HTLV-1 infection showed loss of SHP1 expression and overexpression of CIS, SOCS1, PIAS3. However, this overexpression was not adequate to block constitutive activation of the JAK/STAT pathway. Thus, multiple levels of IL-2 receptor signal deregulation are found in HTLV-1 transformed cells, which may be a result of early loss of SHP1 expression.

Adult↗
PubMed1983

Isolation and partial purification of a new class of transforming growth factors from an avian sarcoma virus-transformed rat cell line.

Transformation of rat cells by avian sarcoma viruses induced the release of growth factors into serum-free conditioned medium. An avian sarcoma virus-transformed rat cell line, 77N1, produced and released a polypeptide growth factor, classified as a transforming growth factor (TGF), which transiently promotes anchorage-dependent BALB3T3 A31 cells to form progressively growing colonies in soft agar. The TGF was isolated and partially purified from an extract of 77N1 cells by ion-exchange chromatography on a diethylaminoethyl Sephacel column followed by ammonium sulfate precipitation. The TGF was assumed to have a molecular weight of 11,000 from gel filtration on Sephadex G-50. This TGF did not compete with epidermal growth factor for binding to cell membrane receptors and was not potentiated by epidermal growth factor. The TGF was trypsin and dithiothreitol sensitive as well as heat and acid labile, indicating that it was different from previously reported TGFs of similar molecular weight and thus belonged to a new class of TGFs.

Animals↗
PubMed1976

Susceptibility of normal and transformed cell lines to cytostatic and cytocidal effects exerted by macrophages.

Activated, nonimmune macrophages exerted profound effects on the proliferation and viability of eukaryotic target cells in vitro. Pronounced macrophage-mediated cytostasis was exerted on every rapidly proliferating cell line examined, irrespective of transformation, species derivation, cell type, or growth characteristics. However, the magnitude of cytostasis effected differed markedly among the 40 cell lines tested. There was no evident correlation between susceptibility to cytostasis and degree of transformation. Transformed cell lines with high and with low malignant attributes were affected equally. A comparable pattern was discerned for cytocidal effects of macrophages, in which the susceptibility of transformed targets was independent of the degree of malignancy.

Cell Line↗
PubMed1991

Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 7 beta-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity, and on cell surface protein expression.

The cytotoxicity of 7 beta-hydroxycholesterol (7 beta-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 microM 7 beta-OHC over a period of 72 h whereas 30 microM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7 beta-OHC added to the cultures at concentrations of 20 microM or 30 microM. Cellular fractionation after incubation with 20 microM or 30 microM 7 beta-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7 beta-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 microM 7 beta-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 microM 7 beta-OHC treatment. Fluorescence anisotropy measurements indicated that 20 microM 7 beta-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 microM 7 beta-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 microM 7 beta-OHC. These findings demonstrate that 7 beta-OHC is lethal to highly proliferating cultured glial cells. The high accumulation of 7 beta-OHC in the plasma membrane and its decrease in fluidity, by themselves, do not seem to be involved in the processes leading to cellular death. However, increase of plasma membrane fragility associated with the decrease of CH/PL, which occurs exclusively in plasma membranes isolated from 7 beta-OHC treated transformed cells together with high 7 beta-OHC uptake, are probably implicated in 7 beta-OHC cytotoxicity. The possibility of an additional action mechanism is discussed.

Animals↗
PubMed1978

Molecular association between transplantation antigens and cell surface antigen in adenovirus-transformed cell line.

A rat cell line (A2T2C4) transformed with adenovirus type 2 elicited cytotoxic T lymphocytes in syngeneic rats. Cytotoxicity was abolished by a rabbit antiserum directed against the major histocompatibility (AgB) antigens and by a syngeneic rat antiserum raised against the virus-transformed cell line. The syngeneic antiserum immunoprecipitated surface proteins with apparent molecular weights of 45,000, 19,000, 17,000, and 12,000 from the A2T2C4 cells but it displayed no reactivity against primary rat fibroblasts and spleen cells. The rabbit antiserum against AgB antigens precipitated a 19,000-dalton component from the A2T2C4 cells which was not observed in primary rat fibroblasts. Sequential immunoprecipitation revealed identity between the major polypeptides recognized by the two antisera. Because the rabbit anti-AgB antigen serum was specific for the transplantation antigen subunits and because the syngeneic rat antiserum against the A2T2C4 cells failed to react with the AgB antigens in normal cells, it is concluded that the 19,000-dalton component is coprecipitated with the AgB antigens. Antisera directed specifically against beta2-microglobulin and the alloantigenic AgB antigen subunit also coprecipitated the 19,000-dalton component. These results indicate that the AgB antigen subunits form a ternary complex with a virus-coded protein on the surface of the virus-transformed A2T2C4 cells. This molecular complex may be recognized by the cytoloytic T lymphocytes

Adenoviruses, Human↗
PubMed1994

Syngeneic lysis of reticuloendotheliosis virus-transformed cell lines transfected with Marek's disease virus genes by virus-specific cytotoxic T cells.

Cell-mediated immune responses against Marek's disease virus (MDV) antigens were examined using reticuloendotheliosis virus (REV)-transformed cell lines of two haplotypes (B19B19 and B13B13). These cell lines were stably transfected with cloned fragments of MDV DNA resulting in the expression of the MDV-specific phosphoprotein pp38. Effector cells were obtained from P2a (B19B19) and S13 (B13B13) chickens at 7 days post inoculation with REV, oncogenic or attenuated serotype 1 MDV (JM-16/O and JM-16/A, respectively), serotype 2 MDV (SB-1), or herpesvirus of turkeys (HVT). Transfection of MDV genes did not influence the expression of Class I major histocompatibility complex antigens. The optimal effector to target cell ratio was determined to be 100:1. REV-sensitized effector cells lysed REV cell lines and REV cell lines transfected with MDV DNA in a syngeneic fashion. Effector cells from chickens inoculated with JM-16/O, JM-16/A, SB-1 or HVT lysed only the syngeneic, transfected cell lines, but not the parent REV cell lines. The percentage specific release caused by the MDV-sensitized effector cells was low, but statistically significant.

Animals↗
PubMed1989

Selective killing of human T cell lymphotropic virus type I-transformed cell lines by a damavaricin Fc derivative.

n-Pentyl ether of damavaricin Fc (n-pentyl DvFc) preferentially killed human T-cell lymphotropic virus type I (HTLV-I)-transformed cell lines. The mechanism of action of the drug was investigated using MT-4 cells. Cytotoxic action was diminished by the removal of n-pentyl DvFc from the culture or by the addition of sulfhydryl compounds such as 2-mercaptoethanol and dithiothreitol. The killing activity of n-pentyl DvFc was also diminished by membrane-acting agents including quinidine and diphenylhydantoin. Influx and subsequent efflux of Ca2+ were observed when either HTLV-I infected (MT-4 cells) or uninfected cells were treated with n-pentyl DvFc. An efflux of K+ was observed in HTLV-I infected MT-4 cells immediately after the exposure of the cells to n-pentyl DvFc. The K+ efflux, however, was not observed in the uninfected T cells. n-Pentyl DvFc seems to act primarily on the cell surface of MT-4 cells, leading to the perturbation of membrane function. The restoration of cell growth, however, is critically dependent on the presence of dithiothreitol and 2-mercaptoethanol, implying a role for a free sulfhydryl group in the killing activity.

Anti-Bacterial Agents↗
PubMed1984

Expression of the E4 gene is required for establishment of soft-agar colony-forming rat cell lines transformed by the adenovirus 12 E1 gene.

Rat 3Y1 cells were transfected with recombinant gARC ( pSV2gpt carrying the adenovirus 12 early region 1 [E1] gene), and focus formation was observed in monolayer cultures after culture of cells in gpt-selective medium (Eagle medium containing 10% fetal calf serum, xanthine, thymidine, aminopterin, and mycophenolic acid) for 10 days, followed by focus formation. Transformed E1Y cell lines were then established from these foci. The E1Y cells were transformed morphologically similarly to cells transformed with intact adenovirus 12 DNA but formed no colonies in soft-agar culture and induced tumors in transplanted rats only after a long incubation period. For the establishment of completely transformed cells, 3Y1 cells were transformed with combinations of gARC , pE3 (pBR322 carrying the adenovirus 12 E3 gene), and gE4 ( pSV2gpt carrying the adenovirus 12 E4 gene) DNA. E1- 3Y cells (3Y1 cells transformed with gARC and pE3 DNA), E1- 4Y cells (3Y1 cells transformed with gARC and gE4 DNA), and E1-3- 4Y cells (3Y1 cells transformed with gARC , pE3 , and gE4 DNA) were established. These transformed cell lines were compared for growth in Eagle medium with 2 or 10% fetal calf serum, colony formation in soft-agar culture, and tumor growth in rats transplanted with the transformed cells. Several transformed cell lines of E1- 4Y and E1-3- 4Y cells showed colony formation in soft-agar culture and abundant expression of the E1B gene. T antigen f was seen by immunofluorescence as flecks in these cells, in which the E4 gene was transcribed, but was not seen in E1Y cells, suggesting that T antigen f was encoded by the E4 gene. The suggestion was confirmed by the observation that T antigen f was detected in COS-1 cells transfected singly with gE4 DNA by immunofluorescence with polyclonal and monoclonal antibodies. Transcription of the E4 gene was confirmed in gE4 -transfected COS-1 cells. T antigen f, one of the E4 gene products, was identified as a polypeptide of molecular weight 11,000 (E4- 11K ) by immunoprecipitation with monoclonal antibodies. The above results also suggest that expression of the E4 gene gives cells the advantage of forming colonies in soft-agar culture. A tendency was noticed for E1B gene expression to be enhanced by E4 gene expression. The relationship between enhancement of colony formation in soft-agar culture and enhancement of E1B gene expression is discussed.

Adenoviruses, Human↗
PubMed1977

Assignment of the integration site for simian virus 40 to chromosome 17 in GM54VA, a human cell line transformed by simian virus 40.

GM54VA human cells transformed by simian virus 40 (SV40) were fused with peritoneal macrophages obtained from three different mouse strains. All 27 hybrid clones studied were positive for SV40 tumor antigen in 100% of their cells and contained human chromosome 17. Human chromosome 17 was the only human chromosome present in five of the hybrid clones. Fusion of GM54VA cells and either thymidine kinase (EC 2.7.1.75)-deficient mouse or Chinese hamster fibroblasts resulted in the growth in hypoxanthine-aminopterin-thymidine medium of hybrid clones positive and negative for SV40 tumor antigen. Counterselection of the hybrid clones positive for tumor antigen in medium containing 5-bromodeoxyuridine resulted in the growth of hybrid cells that were negative for tumor antigen. These experiments indicate that negative for tumor antigen. These experiments indicate that SV40 is integrated in only one of the two parental human chromosomes 17. Because the genome of SV40 has been assigned to human chromosome 7 in two other SV40-transformed human cell lines, at least two different integration sites for SV40 would seem to be present in human cells: one located in human chromosome 7 and the other located in human chromosome 17.

Alleles↗
PubMed1981

Chromosome abnormalities associated with salivary gland epithelial cell lines transformed in vitro and in vivo with evidence of a role for genetic imbalance in transformation.

Chromosomal abnormalities associated with five in vitro-transformed male mouse salivary gland epithelial cell lines were compared with those in three cell lines derived from in vivo-induced tumors. All cell lines were hypotetraploid. Structural chromosome abnormalities were found in all cell lines, but no consistent aberration was detected. Nevertheless, losses of chromosomes 1, 4, 7, 9, and 14 were observed in all of the in vitro-transformed cell lines. With the possible exception of chromosome 1, the same chromosome losses were noted in the in vivo-transformed cell lines. In addition, a consistent feature of both in vitro- and in vivo-transformed cell lines was the presence of double minute chromosomes and homogeneously staining regions. Where both of these chromosome types were present in the same cell line, they were mutually exclusive. The Y chromosome was absent in nearly all of the cell lines. These findings are consistent with the view that, in salivary gland epithelium, the malignant phenotype may result from a genetic imbalance caused by specific chromosome losses from tetraploid cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1979

Tumorigenicity and karyotype of rat embryo cell lines transformed by BK virus.

A rat embryo cell line transformed by BK virus was used to induce tumors in rats. Cell lines were established from these tumors. Other sublines were obtained by in vitro cloning of the parental line. Growth characteristics and karyotypes were compared to the tumorigenicity of these cell lines. The in vitro cloned sublines had a low tumorigenicity. Tumorigenicity of the tumor cell lines varied from high to undetectable. The tumor cell line with the highest tumorigenicity also had the highest saturation density in vitro, but otherwise there was little correlation between tumorigenicity and the in vitro characteristics of the cells. Karyotype analysis was done for two cell lines with high or low tumorigenicity which both had a near-diploid complement of chromosomes. The findings were in agreement with the expression-supression model of Rabinowitz and Sachs (1970). The suppression chromosomes seemed to be confined in group A, the expression chromosomes in group B.

Animals↗
PubMed1980

Chromosomal alterations of rat cell lines transformed by human adenovirus type-12 virion, whole DNA and left-end DNA fragments.

A normal rat cell line, 3Y1-B clone 1-6 (3Y1) and its adenovirus (Ad) type-12-transformed derivatives, W4 (transformed by Ad12 Virion), WY3 (transformed by Ad12 whole DNA), CY1-1 (transformed by the Ad12 EcoRI-C fragment, left 16.5%), GY1-1 (transformed by the Ad12 HindIII-G fragment, left 6.8%) and HY1 (transformed by the Ad12 Acd-H fragment, left 4.7%) were studied cytogenetically. 3Y1 and some of the transformed cell lines (W4, WY3 and GY1-1) were diploid or pseudodiploid, while others (CY1-1 and HY1) were hypotetraploid. A metacentric marker M1 was detected in GY1-1 cells and another marker M2 in W4 and CY1-1 cells at a high frequency. By the Giemsa banding technique, the metacentric markers M1 and M2 from these fully transformed cell lines were identified as isochromosomes derived from 1q (M1) and 3q (M2), respectively. On the other hand, the markers were detected only at a low frequency in incompletely transformed HY1 cells. However, hypersomy in chromosome No. 1 was observed at a high frequency in this cell line. It can be concluded that hypersomy of chromosomes No. 1 or 3 found in transformants and metacentric markers found in complete transformants are characteristic features in rat cells transformed by Ad12.

Adenoviruses, Human↗
PubMed1982

Continuing kappa-gene rearrangement in a cell line transformed by Abelson murine leukemia virus.

A cell line transformed by Abelson murine leukemia virus, called PD, is capable of carrying out kappa-gene rearrangement while growing in culture. Subclones of PD have diverse kappa-gene structures, and some derivatives show evidence of continued joining activity after as many as three subclonings. Analysis of PD sublineages has shown that a rearranged chromosome can undergo secondary kappa-gene rearrangements, producing either a new rearrangement or a deletion of C kappa. Although the PD line actively rearranges its kappa genes, its rearranged heavy-chain genes show little variation, and there is no rearrangement of lambda genes. In PD subclones, DNA fragments representing the reciprocal product of kappa-gene rearrangement are often evident, and they may undergo either further rearrangement or deletion. The implications of multiple rearrangements on a single chromosome and of the maintenance of reciprocal fragments are considered in the context of a model that postulates that the V kappa and J kappa segments are not all organized in the DNA in the same transcriptional direction, leading to inversions rather than deletions during joining.

Abelson murine leukemia virus↗
PubMed1977

Oncogenicity of a nude mouse cell line transformed by a human papovavirus.

Primary cultures of NIH nude mouse (nu/nu) kidney cells were transformed with a human papovavirus (MMV). The transformed cell line expressed T-antigen, and MMV DNA was found to be associated with the cell DNA. When NIH nu/nu mice were inoculated with the transformed cells, they developed tumors at the injection site but failed to generate detectable levels of T-antibody. A control group of nu/+ littermates rejected the tumor inoculum but mounted an antibody response to T-antigen. It was proposed that nude mouse cells may be a suitable system to test oncogenicity of in vitro transformed cells.

Animals↗
PubMed1987

Preferential clustering of viral DNA sequences at or near the site of chromosomal rearrangement in fowl adenovirus type 1 DNA-transformed cell lines.

All six transformants obtained by inoculating fowl adenovirus type 1 (CELO virus) DNA or its fragments into a rat cell line of normal karyotype had more than 50 copy-equivalents of viral DNA sequences. Each of the transformants had almost all if not all of these viral DNA sequences clustered on a marker chromosome(s). Although the marker chromosome(s) differed from one cell line to another, viral DNA sequences preferentially clustered in or near the achromatic (or light-stained) region of the G-banded marker chromosomes where chromosomal rearrangement or translocation occurred. These results indicate that no particular chromosome is required to act as the integration site of viral DNA for the transformation of cells, but chromosomal rearrangement at or near the cluster of viral DNA sequences might contribute to the transformation.

Adenoviridae↗
PubMed1985

Structure of viral DNA in a rat cell line transformed by the cloned EcoRI-C fragment of adenovirus 12.

A DNA segment carrying viral DNA was cloned from a rat cell line transformed by the cloned EcoRI-C fragment (0 to 16.4 map units) of human adenovirus type 12(Ad12), and the viral sequence in the clone was analysed. The cloned segment contained the region from nucleotide positions 118 to 3520 of the Ad12 genome in the middle. No unique structure was found at the viral and non-viral DNA junctions. When examined the transforming activity, the conserved viral sequence was able to transform rat 3Y1 cells efficiently. Southern blotting analysis of the viral sequence in five re-transformed cell lines showed that the viral sequence was inserted at different sites of cellular DNA. These results indicate that (I) the Ad12 DNA moiety from the enhancer-promoter region of the E1A gene to the end of the E1B gene contains enough information for efficient transformation of the rat cell, and (II) integration of the viral sequence at unique cellular sites is not prerequisite for transformation.

Adenoviruses, Human↗