Transformed Hamster Cells Research Articles

Alternative production of Bcl-2 and Bax by tumor cells determines the rates of in vivo tumor progression: Suggested mechanisms

The hypothesis tested in the study suggests that mechanisms of the earlier described delayed or accelerated tumor progression may be regulated by the antiapoptotic and proapoptotic cellular programs activated in stress reactions of transformed cells to the host normal cellular environment. Therefore, spontaneously transformed hamster cell line STHE, its bcl-2-transduced line STHE-Bcl-2, and 64 of their descendant tumor cell variants naturally selected in two in vivo regimes (local tumor growth versus dissemination) were examined. The role of Bcl-2 and the possible activation of endogenous death-signaling Bax, Ras, and HSP90/HSP70 stress proteins in STHE (Bcl-2+/-) tumor cell variants were studied in dynamics of in vivo tumor progression. The data demonstrate: (1) Immediate in vivo activation of Bax and of HSP90/HSP70 stress proteins in disseminated STHE cells on the background of accelerated tumor progression; (2) No immediate activation of Bax and the gradual downregulation of Bcl-2 in STHE-Bcl-2 cells on the background of delayed tumor progression; (3) Alternative and mutually suppressive character of Bcl-2 and Bax expression in both regimes of tumor progression; (4) In the later stages of tumor progression, the regular transit of the initial Bcl-2 antiapoptotic, Bax-suppressing program, and the delayed tumor progression towards Bcl-2 loss, activation of Bax, and acceleration of tumor progression. Thus, the delay of tumor progression is apparently determined by the ability of Bcl-2-expressing tumor cells to extinguish the cell-damaging environmental stress signals and Bax activation, while its acceleration correlates with Bcl-2 loss, activation of proapoptotic Bax, and tumor cells damage.

Demethylation of host-cell DNA at the site of avian retrovirus integration

The transcriptional activity of an integrated retroviral copy strongly depends on the adjacent host-cell DNA at the site of integration. Transcribed DNA loci as well as cis-acting sequences like enhancers or CpG islands usually permit expression of nearby integrated proviruses. In contrast, proviruses residing close to cellular silencers tend to transcriptional silencing and CpG methylation. Little is known, however, about the influence of provirus integration on the target sequence in the host genome. Here, we report interesting features of a simplified Rous sarcoma virus integrated into a non-transcribed hypermethylated DNA sequence in the Syrian hamster genome. After integration, CpG methylation of this sequence has been lost almost completely and hypomethylated DNA permits proviral transcription and hamster cell transformation by the proviral v-src oncogene. This, however, is not a stable state, and non-transformed revertants bearing transcriptionally silenced proviruses segregate with a high rate. The provirus silencing is followed by DNA methylation of both provirus regulatory regions and adjacent cellular sequences. This CpG methylation is very dense and resistant to the demethylation effects of 5-aza-2′-deoxycytidine and/or trichostatin A. Our description exemplifies the capacity of retroviruses/retroviral vectors to overcome, at least transiently, negative position effects of DNA methylation at the site of integration.

Identification of CpG islands hypermethylated in tumor and transformed cells by the methylation-sensitive arbitrarily primed polymerase chain reaction

Carcinogenesis is associated with hypermethylation of certain DNA regions, which can suppress transcription of the corresponding genes. With a version of methylation-sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR), three fragments were identified that are hypermethylated in human cervical carcinomas and transformed hamster cells expressing exogenous h-N-ras. The fragments were cloned, sequenced, and shown to possess properties characteristic of CpG islands. Of these, one was identified as a fragment of the β3A-adaptin gene and two showed no homology to known sequences. MS PCR with specific primers showed that the CpG island of the β3A-adaptin gene was hypermethylated in four (15%) of 27 cervical carcinomas examined. Southern blotting with the cloned hamster DNA fragment confirmed hypermethylation of the corre-sponding CpG island in three hamster cell lines expressing N-ras. The method proved useful for identification of CpG islands, including those hypermethylated in tumor and transformed cells.

Mammalian S-phase checkpoint integrity is dependent on transformation status and purine deoxyribonucleosides.

In eukaryotic cells arrested in S-phase, checkpoint controls normally restrain mitosis until after replication. We have identified an array of previously unsuspected factors that modulate this restraint, using transformed hamster cells in which cycle controls are known to be altered in S-phase arrest. Arrested cells accumulate cyclin B, the regulatory partner of the mitotic p34(cdc2) kinase, which is normally not abundant until late G(2) phase; treatment of arrested cells with caffeine produces rapid S-phase condensation. We show here that such S-phase checkpoint slippage, as visualised through caffeine-dependent S-phase condensation, correlates with rodent origin and transformed status, is opposed by reverse transformation, and is favoured by c-src and opposed by wnt1 overexpression. Slippage is also dependent on a prolonged replicative arrest, and is favoured by arrest with hydroxyurea, which inhibits ribonucleotide reductase. This last is a key enzyme in deoxyribonucleotide synthesis, recently identified as a determinant of malignancy. Addition of deoxyribonucleosides shows that rapid S-phase condensation is suppressed by a novel checkpoint mechanism: purine (but not pyrimidine) deoxyribonucleosides, like reverse transformation, suppress cyclin B/p34(cdc2) activation by caffeine, but not cyclin B accumulation. Thus, ribonucleotide reductase has an unexpectedly complex role in mammalian cell cycle regulation: not only is it regulated in response to cycle progression, but its products can also reciprocally influence cell cycle control kinase activation.

Confirmed identies of proteins from a two-dimensional map of Syrian hamster embryo cells

The Syrian hamster embryo (SHE) cell transformation assay is widely used to screen chemicals for carcinogenic potential. However, the biochemical mechanisms of transformation in SHE cells are incompletely understood relative to other rodent systems. Thus identification of proteins which change during transformation can provide clues to biochemical mechanisms. Previously, we published a map of SHE cell proteins based on comparisons to other maps. In this report we provide direct sequence analysis of numerous proteins which were previously identified solely by electrophoretic mobility. Protein sequencing verified original spot identifications and extended the range of identified proteins. The updated map will assist in evaluating biochemical mechanisms of morphological transformation in hamster cells.

Confirmed identities of proteins from a two-dimensional map of Syrian hamster embryo cells.

The Syrian hamster embryo (SHE) cell transformation assay is widely used to screen chemicals for carcinogenic potential. However, the biochemical mechanisms of transformation in SHE cells are incompletely understood relative to other rodent systems. Thus identification of proteins which change during transformation can provide clues to biochemical mechanisms. Previously, we published a map of SHE cell proteins based on comparisons to other maps. In this report we provide direct sequence analysis of numerous proteins which were previously identified solely by electrophoretic mobility. Protein sequencing verified original spot identifications and extended the range of identified proteins. The updated map will assist in evaluating biochemical mechanisms of morphological transformation in hamster cells.

Simultaneous ESR measurements of the kinetics of oxygen consumption and spin label reduction by mammalian cells

AbstractNitroxide spin labels and probes were applied to measurements of cell redox activities and oxygen concentration by ESR. Current experimental methods often require large numbers of cells and monitoring of only the amplitude of the ESR signal. This can result in experimental artifacts and data misinterpretation. An improved method is described for accurate measurement of nitroxide intensity and linewidth, which in turn provides information about cell redox activities and oxygen concentration. The method is based on fitting of ESR spectra using a fast convolution least‐squares algorithm suitable for inhomogeneously broadened ESR lines. All fitting parameters, including the Lorentzian component of the linewidth and the double integrated intensity, are extracted from the experimental spectra directly. The experiments were carried out at cell concentrations of 5 × 106 cell ml−1 or less to prevent cell crowding and at initial nitroxide concentration of 1 mM or 100 μM to ensure that nitroxide metabolism by the cells was not diffusion limited. Examples include experiments with cultured baby hamster kidney and adenovirus transformed hamster cells using the nitroxide radicals TEMPOL (2,2,6,6‐tetramethylpiperidine‐N‐oxyl‐4‐ol) and 15N‐labeled perdeuteriated TEMPONE (15NPDT) (2,2,6,6‐tetramethylpiperidine‐N‐oxyl‐4‐one). Under these experimental conditions, the rates of nitroxide metabolism derived from changes in nitroxide intensity are independent of oxygen concentration.

Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments.

Insertion of foreign DNA into an established mammalian genome can extensively alter the patterns of cellular DNA methylation. Adenovirus type 12 (Ad12)-transformed hamster cells, Ad12-induced hamster tumor cells, or hamster cells carrying integrated DNA of bacteriophage lambda were used as model systems. DNA methylation levels were examined by cleaving cellular DNA with Hpa II, Msp I, or Hha I, followed by Southern blot hybridization with 32P-labeled, randomly selected cellular DNA probes. For several, but not all, cellular DNA segments investigated, extensive increases in DNA methylation were found in comparison with the methylation patterns in BHK21 or primary Syrian hamster cells. In eight different Ad12-induced hamster tumors, moderate increases in DNA methylation were seen. Increased methylation of cellular genes was also documented in two hamster cell lines with integrated Ad12 DNA without the Ad12-transformed phenotype, in one cloned BHK21 cell line with integrated plasmid DNA, and in at least three cloned BHK21 cell lines with integrated lambda DNA. By fluorescent in situ hybridization, the cellular hybridization probes were located to different hamster chromosomes. The endogenous intracisternal A particle genomes showed a striking distribution on many hamster chromosomes, frequently on their short arms. When BHK21 hamster cells were abortively infected with Ad12, increases in cellular DNA methylation were not seen. Thus, Ad12 early gene products were not directly involved in increasing cellular DNA methylation. We attribute the alterations in cellular DNA methylation, at least in part, to the insertion of foreign DNA. Can alterations in the methylation profiles of hamster cellular DNA contribute to the generation of the oncogenic phenotype?

Lymphotropic papovavirus transforms hamster cells without altering the amount or stability of p53

Expression of the early regions of several primate polyomaviruses (SV40, BKV, JCV, and LPV) in hamster cells induces transformation, manifested by the ability to grow in soft agar. Hamster cells transformed by SV40 contain complexes between the SV40 T antigen and the cellular tumor suppressor protein p53. We detected analogous complexes between p53 and the BKV T antigen in hamster cells transformed by the BKV early region, where the half life of p53 increased 16-fold. However, neither a LPV-transformed hamster fibroblast cell line [LPV-HE (F); K. K. Takemoto and T. Kanda, 1984, J. Virol. 50, 100–105] nor BHK-21 cells transformed by the LPV early region contained detectable complexes between the LPV T antigen and p53, nor was the stability of p53 in LPV transformed BHK-21 cells altered. Association between hamster p53 and the LPV T antigen expressed as glutathione S-transferase fusion protein could not be detected in vitro. These data indicate that alteration of the amount or stability of p53 is not required for transformation of hamster cells by LPV. However, as viruses such as SV40 and BKV whose T antigens bind p53 are oncogenic in hamsters, whereas LPV is not, the alteration of p53 amount or stability may be required for tumorigenesis.

Alterations in the levels of expression of specific cellular genes in adenovirus-infected and -transformed cells

There is increasing evidence that changes in the transcriptional program of cellular genes in virus-transformed cells can contribute to virus transformation. It is, therefore, important to study altered expression patterns of cellular genes in adenovirus-infected and -transformed cells. We have used 40 different cellular genes or gene segments as hybridization probes to analyze the cytoplasmic RNA from adenovirus type 2 (Ad2)-infected KB cells, from Ad5-transformed human cells (293) or from several Ad2- or adenovirus type 12 (Ad12)-transformed hamster cell lines. Many of the genes probed were not expressed in human or hamster cells. Transcription of the ADPRT and the heat shock protein 70 genes was increased in Ad2-infected KB cells and in 293 cells. In Ad2-infected KB cells, c- myc gene transcription was decreased. In 293 cells and in three adenovirus-transformed hamster cell lines (T637, BHK21-Ad2E1A-E1B, and BHK21-Ad2 HindIII-G), the transcription of the c- jun gene was increased, whereas c- myc transcription was decreased in the latter two cell lines. The data presented here demonstrate that, among 40 different mammalian gene probes, alterations in steady state levels of RNA were detected for five of these genes. These results suggest major alterations in transcription patterns in adenovirus-infected and -transformed cells.

Altered expression of adenovirus 12 DNA-binding protein but not DNA polymerase during abortive infection of hamster cells

Replication of human adenovirus type 12 DNA is blocked in abortively infected baby hamster kidney cells. The activity and accumulation of adenovirus 12 DNA polymerase is equivalent in infected hamster and human cell extracts. However, the accumulation of adenovirus type 12 DNA-binding protein is approximately 120-fold lower in extracts from infected hamster cells when compared to infected permissive human cells. This difference in accumulation is not due to replication of viral DNA during productive infection, since this difference is observed in the presence of hydroxyurea. The DNA-binding protein from infected hamster cells retains the ability to bind denatured DNA-cellulose. An adenovirus 5 early region 1 transformed hamster cell line competent to complement the adenovirus 12 DNA replication defect also stimulates accumulation of the DNA-binding protein even when the cells are treated with hydroxyurea. Thus, the reduced expression of the viral DNA-binding protein may play a role in the mechanism of abortive infection of hamster cells by adenovirus 12.

BK virus early RNA transcripts in stably transformed cells: enhanced levels induced by dibutyryl cyclic AMP, forskolin and 12-O-tetradecanoylphorbol-13-acetate treatment

The stably BK virus (BKV)-transformed hamster cell line BKT-1B and control BHK-21 cells were treated with dibutyryl cAMP (bu2cAMP), the adenylate cyclase activator forskolin, and the tumour-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). Cultures were stimulated for 30 min (short) and for 24 h (long). Northern blot analysis showed that for bu2cAMP and TPA both short and long stimulation resulted in significant increases in the levels of BKV early transcripts. Short exposure to forskolin resulted in a moderate increase and long exposure in a definite increase. In all cases the increased levels were maintained for at least 24 h after short stimulation was stopped. Experiments including the transcription inhibitor actinomycin D revealed that the enhanced levels of early BKV expression after treatment with the stimuli were due to induced RNA synthesis rather than to stabilization of the RNA. No DNA amplification of the early BKV sequences could be detected in the induced cells. The results are discussed with regard to possible roles for a cAMP-responsive element and a phorbol ester-responsive element, shown by sequencing to be present in the control region of the integrated BKV genome of the BKT-1B cells.

The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification

Herpes simplex virus (HSV) induces within the host cell genome DNA amplification which can be suppressed by coinfection with adeno-associated virus (AAV). To characterize the AAV functions mediating this effect, cloned AAV type 2 wild-type or mutant genomes were transfected into simian virus 40 (SV40)-transformed hamster cells together with the six HSV replication genes (encoding UL5, UL8, major DNA-binding protein, DNA polymerase, UL42, and UL52) which together are necessary and sufficient for the induction of SV40 DNA amplification (R. Heilbronn and H. zur Hausen, J. Virol. 63:3683-3692, 1989). The AAV rep gene was identified as being responsible for the complete inhibition of HSV-induced SV40 DNA amplification. Likewise, rep inhibited origin-dependent HSV replication. rep neither killed the transfected host cells nor interfered with gene expression from the cotransfected amplification genes. This points to a specific interference with HSV-induced DNA amplification.

Herpes simplex virus-induced “Rolling circle” amplification of SV40 DNA sequences in a transformed hamster cell line correlates with tandem integration of the SV40 genome

Infection with herpes simplex virus leads to amplification of SV40 DNA in various SV40-transformed cells. In earlier studies with the SV40-transformed hamster cell line Elona two different types of DNA amplification could be identified: (i) Bidirectional overreplication of chromosomally integrated SV40 DNA expanding into the flanking cellular sequences (“onion skin” type) and (ii) highly efficient synthesis of extremely large head-to-tail concatemers containing exclusively SV40 DNA (“rolling circle” type). These investigations have indicated that the chromosomally integrated form of SV40 might be the substrate for both types of overreplication. There still had been uncertainties as to whether and how these events were connected. A hypothetical assumption of a recombinational event leading to the excision of SV40 DNA molecules is supported by the results presented here: In this study cloned Elona cell lines were investigated for their ability to amplify SV40 sequences and for the mechanism of amplification utilized. SV40 integration in a partial tandem manner correlates with a strong rolling circle amplification. In contrast, in one cell line harboring a truncated SV40 genome, amplification appears mainly restricted to intrachromosomal bidirectional overreplication. Possible implications for HSV functions involved in the amplification process will be discussed.

A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome

Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of six HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensible for SV40 DNA amplification. Our results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.

Defect of adenovirus type 12 replication in hamster cells: absence of transcription of viral virus-associated and L1 RNAs.

The nonpermissive interaction of hamster cells with human adenovirus type 12 (Ad12) is characterized by a total block of Ad12 DNA replication and late transcription, whereas most of the early functions of Ad12 DNA can be transcribed. Ad2 can replicate in hamster cells. The replication and late transcription defects of Ad12 DNA can be complemented to a certain extent by the E1B functions of Ad2 DNA. This complementation fails, however, to lead to the synthesis of the late Ad12 proteins and to the assembly of infectious virions. It will now be demonstrated that the Ad12 L1 (late genes of group 1) and virus-associated (VA) RNAs are not transcribed in hamster cells. Synthesis of these RNAs in productively infected human cells or Ad2-infected hamster cells is readily detectable by S1 nuclease protection experiments and Northern (RNA) blotting. Similarly, the Ad2-transformed hamster cell line BHK-Ad2E1 fails to complement L1 and VA RNA syntheses after superinfection with Ad12. However, Ad12 infection of the Ad5-transformed hamster cell line BHK297-C131 leads to the transcription of the Ad12 L1 and VA segments. This difference in complementation by the two transformed hamster cell lines might be accounted for by functions in the segment of Ad5 DNA extending between map units 30 and 40 and persisting in the Ad5-transformed hamster cells or by hamster host cell functions which might be operative in cell line BHK297-C131 but not in BHK-Ad2E1 or BHK-21 hamster cells.

Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation

In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene.

Arrangement and expression of integrated adenovirus type 12 DNA in the transformed hamster cell line HA12/7: amplification of Ad12 and c-myc DNAs and evidence for hybrid viral-cellular transcripts

In the genome of the adenovirus type 12 (Ad12)-transformed hamster cell line HA12/7 about three copies of the viral DNA are fixed by integration. The results of blot-hybridization, molecular cloning, and nucleotide sequencing experiments suggest a model for the arrangement of Ad12 DNA molecules in which the left hand terminus of one of the Ad12 DNA copies is linked to unique hamster DNA. The right hand end of this DNA molecule is fused to an inverted copy of a left terminal ∼ 4.3 kb fragment of Ad12 DNA. This ensemble is followed by the second Ad12 DNA copy whose right terminus is again joined to an inverted, supernumerary left terminal ∼ 4.3 kb Ad12 DNA fragment. There is a third Ad12 DNA copy whose right terminus is linked to cellular DNA. In this sequence arrangement, the left terminus of Ad12 DNA is overrepresented, as had been shown earlier (S. Stabel W. Doerfler and R.R. Friis (1980) J. Virol. 36, 22–40). In the presented model, cellular DNA sequences are interspersed in between the three copies of Ad12 DNA. In the left terminus of the integrated Ad12 DNA, transcription of RNA is initiated which extends out into cellular DNA. The interviral DNA junctions are also transcribed The c-myc gene in cell line HA12/7 is amplified about 10-fold and considerably more c-myc RNA has been identified in the Ad12-transformed cells than in BHK21 or in LSH hamster cells. It has been shown previously that the E1 region of Ad12 DNA is transcribed into mRNA in HA12/7 cells (Ortin et al. (1976) J. Virol. 20, 355–372). It remains to be investigated whether c-myc amplification and expression are related to the transformed phenotype of HA12/7 cells.

Absence of BK virus sequences in transformed hamster cells transfected by human tumor DNA

In an attempt to gain insight into the mechanism of oncogenic transformation by BK virus (BKV), a human papovavirus, we have probed for BKV sequences in transformed hamster cells in which oncogenic transformation had occurred as a result of transfection by human tumor DNA positive for BKV sequences. Even though the sources of the transfecting DNA contained BKV sequences, the transformed hamster cells which arose from the transfection for the most part did not retain BKV sequences. In only one barely detectable case was BKV-specific DNA found associated with chromosomal DNA, and in only a small minority of the transformed cells was BKV DNA detected in the Hirt supernatant, indicating an episomal configuration. Even in these few cases where BKV sequences were present in an episomal form, altered migration on gels of some BKV-positive bands (compared to bands derived from cloned viral DNA) suggested deletions and rearrangements of BKV DNA. We employed several different probe methodologies for these studies, including nick-translation, random primer and a non-isotopic biotinylated probe which gave a sensitivity that could detect better than 0.01 copy of viral genome per diploid cell. We conclude that transformation by transfection with human tumor DNA does not require persistence of the BKV viral genome, suggesting that either BKV virus was irrelevant to original oncogenesis in the human tumors, or that BKV employs a ‘hit and run’ mechanism of oncogenesis, in analogy with models proposed by others for herpesvirus oncogenesis.

Expression of hamster MHC class I antigens in transformed cells and tumours induced by human adenoviruses

The steady-state levels of hamster MHC class I mRNA and cell surface protein were analysed in cells transformed by either adenovirus type 12 (AD12) or types 2 or 5 (Ad2 or Ad5). All cell lines were oncogenic in new born and adult hamsters. A great reduction in both class I mRNA and protein was observed in AD12 transformed cells compared to cells transformed by Ad2 or Ad5. Analysis of class I mRNA in solid tumours induced in hamsters by Ad transformed cell lines also showed greatly reduced mRNA levels in tumours induced by Ad12 compared to those induced by Ad2 or Ad5. This suggests that, in the Ad transformed hamster cell system, reduction in the level of MHC class I gene expression is not necessarily associated with tumour formation.