Hamster Cell Line Research Articles

Induction of aneuploidy by nickel sulfate in V79 Chinese hamster cells

The ability of nickel sulfate (NiSO 4) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100–400 μM NiSO 4 for 24 h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay. Aneuploid cells were induced in a significant fraction of the cell population 24–48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO 4. The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose. These results indicate that NiSO 4 has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.

Foreign DNA Integration: GENOME-WIDE PERTURBATIONS OF METHYLATION AND TRANSCRIPTION IN THE RECIPIENT GENOMES

In hamster cells transgenic for the DNA of adenovirus type 12 (Ad12) or for the DNA of bacteriophage lambda, the patterns of DNA methylation in specific cellular genes or DNA segments remote from the site of transgene insertion were altered. In the present report, a wide scope of cellular DNA segments and genes was analyzed. The technique of methylation-sensitive representational difference analysis (MS-RDA) was based on a subtractive hybridization protocol after selecting against DNA segments that were heavily methylated and hence rarely cleaved by the methylation-sensitive endonuclease HpaII. The MS-RDA protocol led to the isolation of several cellular DNA segments that were indeed more heavily methylated in lambda DNA-transgenic hamster cell lines. By applying the suppressive subtractive hybridization technique to cDNA preparations from nontransgenic and Ad12-transformed or lambda DNA-transgenic hamster cells, several cellular genes with altered transcription patterns were cloned from Ad12-transformed or lambda DNA-transgenic hamster cells. Many of the DNA segments with altered methylation, which were isolated by a newly developed methylation-sensitive amplicon subtraction protocol, and cDNA fragments derived from genes with altered transcription patterns were identified by their nucleotide sequences. In control experiments, no differences in gene expression or DNA methylation patterns were detectable among individual nontransgenic BHK21 cell clones. In one mouse line transgenic for the DNA of bacteriophage lambda, hypermethylation was observed in the imprinted Igf2r gene in DNA from heart muscle. Two mouse lines transgenic for an adenovirus promoter-indicator gene construct showed hypomethylation in the interleukin 10 and Igf2r loci. We conclude that the insertion of foreign DNA into an established mammalian genome can lead to alterations in cellular DNA methylation and transcription patterns. It is conceivable that the genes and DNA segments affected by these alterations depend on the site(s) of foreign DNA insertion.

Transcription coupled repair deficiency results in increased chromosomal aberrations and apoptotic death in the UV61 cell line, the Chinese hamster homologue of Cockayne’s syndrome B

Transcription coupled repair (TCR), a special sub-pathway of nucleotide excision repair (NER), removes transcription blocking lesions rapidly from the transcribing strand of active genes. In this study, we have evaluated the importance of the TCR pathway in the induction of chromosomal aberrations and apoptosis in isogenic Chinese hamster cell lines, which differ in TCR efficiency. AA8 is the parental cell line, which is proficient in the genome overall repair of UV-C radiation induced 6-4 photoproducts (6-4 PP) and the repair of cyclobutane pyrimidine dimer (CPD) from the transcribing strand of active genes. UV61 cells (hamster homologue of human Cockayne’s syndrome (CS) group B cells) originally isolated from AA8, exhibit proficient repair of 6-4 PP but are deficient in CPD removal by the TCR pathway. Upon UV-C irradiation of cells in G1-phase, UV61 showed a dramatic increase in apoptotic response as compared to AA8 cells. Abolition of TCR by treatment with α-amanitin (an inhibitor of RNA polymerase II) in AA8 cells also resulted in an elevated apoptotic response like that observed in UV61 cells treated with UV alone. This suggests that the lack of TCR is largely responsible for increased apoptotic response in UV61 cells. Furthermore, the chromosomal aberrations and sister chromatid exchange (SCE) induced by UV were also found to be higher in UV61 cells than in TCR proficient AA8 cells. This study shows that the increased chromosomal aberrations and apoptotic death in UV61 cells is due to their inability to remove CPD from the transcribing strand of active genes and suggests a protective role for TCR in the prevention of both chromosomal aberrations and apoptosis induced by DNA damage. Furthermore, flow cytometry analysis and time-course appearance of apoptotic cells suggest that the conversion of UV-DNA damage into chromosomal aberrations precedes and determines the apoptotic process.

Codominance associated with overexpression of certain XPD mutations

Mutations in the XPD gene are associated with three complex clinical phenotypes, namely xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (XP-CS), and trichothiodystrophy (TTD). XP is caused by a deficiency in nucleotide excision repair (NER) that results in a high risk of skin cancer. TTD is characterized by severe developmental and neurological defects, with hallmark features of brittle hair and scaly skin, and sometimes has defective NER. We used CHO cells as a system to study how specific mutations alter the dominant/recessive behavior of XPD protein. Previously we identified the T46I and R75W mutations in two highly UV-sensitive hamster cell lines that were reported to have paradoxically high levels of unscheduled DNA synthesis. Here we report that these mutants have greatly reduced XPD helicase activity and fully defective NER in a cell-extract excision assay. We conclude that the unscheduled DNA synthesis seen in these mutants is caused by abortive “repair” that does not contribute to cell survival. These mutations, as well as the K48R canonical helicase-domain mutation, each produced codominant negative phenotypes when overexpressed in wild-type CHO cells. The common XP-specific R683W mutation also behaved in a codominant manner when overexpressed, which is consistent with the idea that this mutation may affect primarily the enzymatic activity of the protein rather than impairing protein interactions, which may underlie TTD. A C-terminal mutation uniquely found in TTD (R722W) was overexpressed but not to levels sufficiently high to rigorously test for a codominant phenotype. Overexpression of mutant XPD alleles may provide a simple means of producing NER deficiency in other cell lines.

The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal locations and requires specific DNA sequence elements for activity.

To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-beta) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-beta in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-beta region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-beta deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-beta region is sufficient to direct initiation and that specific DNA sequences in the ori-beta region are required for efficient initiation activity.

Efficient male and female germline transmission of a human chromosomal vector in mice.

A small accessory chromosome that was mitotically stable in human fibroblasts was transferred into the hprt(-) hamster cell line CH and developed as a human chromosomal vector (HCV) by the introduction of a selectable marker and the 3' end of an HPRT minigene preceded by a loxP sequence. This HCV is stably maintained in the hamster cell line. It consists mainly of alphoid sequences of human chromosome 20 and a fragment of human chromosome region 1p22, containing the tissue factor gene F3. The vector has an active centromere, and telomere sequences are lacking. By transfecting a plasmid containing the 5' end of HPRT and a Cre-encoding plasmid into the HCV(+) hamster cell line, the HPRT minigene was reconstituted by Cre-mediated recombination and expressed by the cells. The HCV was then transferred to male mouse R1-ES cells and it did segregate properly. Chimeras were generated containing the HCV as an independent chromosome in a proportion of the cells. Part of the male and female offspring of the chimeras did contain the HCV. The HCV(+) F1 animals harbored the extra chromosome in >80% of the cells. The HCV was present as an independent chromosome with an active centromere and the human F3 gene was expressed from the HCV in a human-tissue-specific manner. Both male and female F1 mice did transmit the HCV to F2 offspring as an independent chromosome with properties similar to the original vector. This modified small accessory chromosome, thus, shows the properties of a useful chromosomal vector: It segregates stably as an independent chromosome, sequences can be inserted in a controlled way and are expressed from the vector, and the HCV is transmitted through the male and female germline in mice.

Gene amplification in fibroblasts from ataxia telangiectasia (AT) patients and in X-ray hypersensitive AT-like Chinese hamster mutants.

In search of functions involved in the regulation of gene amplification, and given the relevance of chromosome breakage in initiating the process, we analyzed the gene amplification ability of cells hypersensitive to inducers of DNA double-strand breaks and defective in cell cycle control: two human fibroblast strains derived from patients affected by ataxia telangiectasia (AT) and two hamster mutant cell lines belonging to complementation group XRCC8 of the rodent X-ray-sensitive mutants. These mutants are considered hamster models of AT cells. To measure gene amplification, the frequency and the rate of occurrence of N-(phosphonacetyl)-L-aspartate resistant cells were determined. In both hamster mutants, these two parameters were increased by about an order of magnitude compared with parental cells, suggesting that amplification ability was increased by the genetic defect. In primary AT fibroblasts, as in normal human fibroblasts, gene amplification was undetectable and a block in the G(1) phase of the cell cycle was induced upon PALA treatment. These results suggest that in AT fibroblasts, where only the ATM gene is mutated, ATM-independent mechanisms prevent gene amplification, while, in the immortalized hamster cell lines, which are already permissive for gene amplification, the AT-like defect increases the probability of gene amplification.

Overexpression of the DNA-binding domain of poly(ADP-ribose) polymerase inhibits rejoining of ionizing radiation-induced DNA double-strand breaks

Purpose : To assess the influence of trans -dominant inhibition of poly(ADP-ribosyl)ation on the rejoining kinetics of radiation-induced DNA double-strand breaks (DSB). Materials and methods : Stable transfectants of the SV40-transformed hamster cell line CO60 were used: COM3 cells contain a construct to overexpress the poly(ADP-ribose) polymerase (PARP-1) DNA-binding domain (DBD) when induced by dexamethasone, as well as a construct for the constitutive overexpression of the human glucocorticoid receptor (Hg0). COR3 are control cells containing only the Hg0 plasmid. DSB induction and rejoining in X-irradiated cells was assessed by DNA pulsed-field electrophoresis. Results : DSB induction was identical in both cell lines and independent of the presence of dexamethasone. DSB rejoining kinetics was independent of dexamethasone in COR3 cells and identical to COM3 cells without dexamethasone. However, in COM3 cells treated with dexamethasone to induce PARP-1 DBD overexpression, the fast component of the rejoining kinetic was largely reduced, and residual fragmentation increased concomitant with the increased damage fraction in slow rejoining. Conclusions : The results indicate that inhibition of cellular PARP-1 does not affect the rate-limiting step of either fast or slow DSB rejoining. Rather, it appears that absence of poly(ADP-ribosyl)ation due to dominant negative PARP-1 expression induces a shift from rapid to slow DSB rejoining and by this mechanism PARP inhibition may increase the risk of repair failures.

Suppression of the radiation-sensitive phenotype of hamster irs1 and irs2 strains selected for resistance to 3-aminobenzamide

Purpose : The radiosensitive hamster cell lines irs1 and irs2 have phenotypic similarities to cells defective in the early response to DNA-damaging agents as a result of mutations of the genes encoding poly(ADP-ribose)polymerase (PARP) or ataxia-telangiectasia mutated (ATM). Whether modification of PARP activity through selection of strains resistant to 3-aminobenzamide (3-AB) would affect the radiosensitive phenotype of irs1 and irs2 was examined. Materials and methods : 3-AB-resistant strains of irs1, irs2 and their parent line V79 were established and their sensitivity to DNA-damaging agents was measured. In some 3-AB-resistant strains, the radiation resistance of DNA synthesis and the induction of apoptosis were also assayed. Additionally, a number of aspects of PARP function were measured. Results : Independently selected 3-AB-resistant strains of irs2 showed nearly complete suppression of radiation sensitivity, sensitivity to topoisomerase inhibitors, and radioresistant DNA synthesis. 3-AB-resistant strains of irs1 showed partial suppression of phenotype while 3-AB-resistant strains of V79 had no sensitivity changes. The induction of apoptosis in 3-AB-resistant strains of irs2 required substantially higher radiation doses than for irs2 itself. 3-AB-resistant strains had no detectable alteration of PARP level or cleavage following ionizing irradiation and there were no mutations in the PARP gene. Conclusions : Suppression of radiosensitivity associated with 3-AB resistance has important implications for mechanisms of tolerance to damage because it is able to override responses associated with specific genetic defects.

Characterization of Cell-Surface Determinants Important for Baculovirus Infection

Baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. To investigate the cell-surface molecules important for infection of baculovirus into mammalian cells, we constructed a recombinant baculovirus, Ac64-CAluc, which has gp64 and luciferase genes under the polyhedrin and the CAG promoter, respectively. For controls, we constructed recombinant viruses possessing vesicular stomatitis virus (VSV) G protein, mouse hepatitis virus (MHV) S protein, or green fluorescent protein (GFP) gene under the polyhedrin promoter and the luciferase gene under the CAG promoter (AcVSVG-CAluc, AcMHVS-CAluc, and AcGFP-CAluc). Treatment of HepG2 cells with phospholipase C markedly reduced the reporter gene expression by Ac64-CAluc or AcVSVG-CAluc in a dose-dependent manner, whereas AcMHVS-CAluc was shown to be resistant to the treatment. Inhibition with purified lipids and susceptibility to the mutant CHO hamster cell lines deficient in phospholipids synthesis suggest that the interaction of gp64 and phospholipids on the cell surface might play an important role in baculovirus infection into mammalian cells.

Transcription-coupled repair is inducible in hamster cells.

In mammalian cells, the rate of nucleotide excision repair of UV dimers is heterogeneous throughout the genome, with repair occurring more rapidly in the transcribed strand of active genes than in the genome overall. This repair pathway is termed transcription-coupled repair (TCR) and is thought to permit the rapid resumption of RNA synthesis following UV irradiation. To evaluate the inducibility of the TCR process, we examined the repair of UV-induced cyclobutane pyrimidine dimers (CPDs) at the level of the gene following exposure of hamster cells to a sub-lethal UV fluence, 3 h prior to a higher dose. Repair was detected by a well-established technique allowing quantification of CPDs at the level of a specific strand by Southern blot hybridization. Here, we show that prior low-dose irradiation clearly enhanced the early rate of CPD removal in the transcribed strand of the active DHFR gene. Furthermore, the RNA synthesis recovery following UV exposure was stimulated by the priming UV dose. Thus, we provide evidence for an inducible TCR response to CPDs in hamster cells. This pathway is independent of the p53 activation, since the hamster cell line that we used expresses high levels of mutant p53 protein.

Multiple blocks to human immunodeficiency virus type 1 replication in rodent cells.

The recent identification of human gene products that are required for early steps in the human immunodeficiency virus type 1 (HIV-1) life cycle has raised the possibility that rodents might be engineered to support HIV-1 infection. Therefore, we have examined the ability of modified mouse, rat, and hamster cell lines to support productive HIV-1 replication. Rodent cells, engineered to support Tat function by stable expression of a permissive cyclin T1 protein, proved to be able to support reverse transcription, integration, and early gene expression at levels comparable to those observed in human cell lines. Surprisingly, however, levels of CD4- and coreceptor-dependent virus entry were reduced to a variable but significant extent in both mouse and rat fibroblast cell lines. Additional posttranscriptional defects were observed, including a reduced level of unspliced HIV-1 genomic RNA and reduced structural gene expression. Furthermore, the HIV-1 Gag precursor is generally inefficiently processed and is poorly secreted from mouse and rat cells in a largely noninfectious form. These posttranscriptional defects, together, resulted in a dramatically reduced yield of infectious virus (up to 10,000-fold) over a single cycle of HIV-1 replication, as compared to human cells. Interestingly, these defects were less pronounced in one hamster cell line, CHO, which not only was able to produce infectious HIV-1 particles at a level close to that observed in human cells, but also could support transient, low-level HIV-1 replication. Importantly, the blocks to infectious virus production in mouse and rat cells are recessive, since they can be substantially suppressed by fusion with uninfected human cells. These studies imply the existence of one or more human gene products, either lacking or nonfunctional in most rodent cells that are critical for infectious HIV-1 virion morphogenesis.

X-ray-induced damage repair in exponentially growing and growth arrested confluent poly(adenosine diphosphate-ribose) polymerase-deficient V79 chinese hamster cell line.

We studied the role of PARP in X-ray-induced damage repair using V79 Chinese hamster cells and two derivative cell lines ADPRT54 and ADPRT351 deficient in poly(adenosine diphosphate-ribose) polymerase (PARP) activity. We previously demonstrated that these PARP-deficient cells had drastically reduced levels of p53. Further, these cells were also deficient in downstream endpoints of p53 signaling. In the present study we showed that exponentially growing ADPRT54 and ADPRT351 were hypersensitive to X-radiation compared to the parental V79 cells. Under this condition of growth, although the parental V79 cells exhibit G1 arrest in response to X-irradiation, the PARP-deficient cells do not undergo this specific p53-dependent cell cycle arrest. In contrast, all the cell lines showed similar sensitivity to X-radiation under growth arrested conditions. Further, all the cell lines were equally proficient in performing potentially lethal damage repair (PLDR). Our findings suggest that: i) PARP is involved in X-ray-induced damage repair in replicating cells; ii) PARP is not required for X-ray-induced damage repair in quiescent cells; iii) PARP does not participate in PLDR; iv) deficiency of PARP may potentiate the cytotoxicity of X-irradiation by interfering with the p53-dependent G1 block that occurs after X-irradiation. These results suggest the intriguing possibility that the approach of inhibition of PARP combined with X-radiation may have therapeutic potential for the treatment of fast growing tumors. However, this approach may not be beneficial for slow growing/quiescent tumors.

Concurrent detection of gene mutations and chromosome aberrations induced by five chemicals in a CHL/IU cell line incorporating a gpt shuttle vector

We previously established a transgenic Chinese hamster CHL/IU cell line, designated as KN63, for concurrent analysis of gene mutations and chromosome aberrations. The KN63 cell line contains copies of a shuttle vector with the Escherichia coli gpt gene as a mutational target in its chromosome. To evaluate the sensitivity of the cell line to various types of mutagens, methyl methanesulfonate (MMS), N-ethyl- N-nitrosourea (ENU), mitomycin C (MMC), vincristine sulfate (VIN) and C.I. basic red 9 hydrochloride (CIB) were assayed. KN63 cells were treated with each test chemical and gene mutations were detected in the gpt gene of the shuttle vector rescued from the KN63 cell genome into an E. coli host. Chromosome aberrations were concurrently evaluated by conventional metaphase analysis. MMS, ENU and MMC induced both gene mutations and structural chromosome aberrations in KN63 cells, with more efficient induction of the latter. VIN, a well-known aneugen, produced only numerical changes to chromosomes, while CIB was negative for both types of alteration. KN63 cells were as sensitive to MMS, ENU, MMC and VIN as Chinese hamster cell lines such as CHL, CHO and V79 cells. The characteristics of test chemicals indicated by this system should be useful for understanding endpoints in chemical mutagenesis.

Cellular and species resistance to murine amphotropic, gibbon ape, and feline subgroup C leukemia viruses is strongly influenced by receptor expression levels and by receptor masking mechanisms.

Chinese hamster ovary (CHO) cells are resistant to infections by gibbon ape leukemia virus (GALV) and amphotropic murine leukemia virus (A-MLV) unless they are pretreated with tunicamycin, an inhibitor of N-linked glycosylation. These viruses use the related sodium-phosphate symporters Pit1 and Pit2, respectively, as receptors in nonhamster cells, and evidence has suggested that the corresponding transporters of CHO cells may be masked by tunicamycin-sensitive secreted inhibitors. Although the E36 line of Chinese hamster cells was reported to secrete the putative Pit2 inhibitor and to be sensitive to the inhibitory CHO factors, E36 cells are highly susceptible to both GALV and A-MLV in the absence of tunicamycin. Moreover, expression of E36 Pit2 in CHO cells conferred tunicamycin-independent susceptibilities to both viruses. Based on the latter results, it was suggested that E36 Pit2 must functionally differ from the endogenous Pit2 of CHO cells. To test these ideas, we analyzed the receptor properties of CHO Pit1 and Pit2 in CHO cells. Surprisingly, and counterintuitively, transfection of a CHO Pit2 expression vector into CHO cells conferred strong susceptibility to both GALV and A-MLV, and similar overexpression of CHO Pit1 conferred susceptibility to GALV. Thus, CHO Pit2 is a promiscuous functional receptor for both viruses, and CHO Pit1 is a functional receptor for GALV. Similarly, we found that the natural resistance of Mus dunni tail fibroblasts to subgroup C feline leukemia viruses (FeLV-C) was eliminated simply by overexpression of the endogenous FeLV-C receptor homologue. These results demonstrate a novel and simple method to unmask latent retroviral receptor activities that occur in some cells. Specifically, resistances to retroviruses that are caused by subthreshold levels of receptor expression or by stoichiometrically limited masking or interference mechanisms can be efficiently overcome simply by overexpressing the endogenous receptors in the same cells.

An alternate form of Ku80 is required for DNA end-binding activity in mammalian mitochondria.

Mammalian mitochondrial DNA end-binding activity is nearly indistinguishable from that of nuclear Ku. This observation led to the hypothesis that mitochondrial DNA end-binding activity is in part dependent upon Ku80 gene expression. To test this hypothesis, we assayed for Ku activity in mitochondrial extracts prepared from the xrs-5 hamster cell line that lacks Ku80 mRNA expression. Mitochondrial protein extracts prepared from this cell line lacked the DNA end-binding activity found in similar extracts prepared from wild-type cells. Azacytidine-reverted xrs-5 cells that acquired nuclear DNA end-binding activity also acquired mitochondrial DNA end-binding activity. Western blot analysis of human mitochondrial protein extracts using a monoclonal antibody specific for an N-terminal epitope of Ku80 identified a protein with an apparent molecular weight of 68 kDa. This mitochondrial protein was not detected by a monoclonal antibody specific for an epitope at the C-terminal end of Ku80. Consistently, while both the N- and C-terminal Ku80 monoclonal antibodies supershifted the nuclear DNA end-binding complex on an electrophoretic mobility shift assay, only the N-terminal monoclonal antibody supershifted the mitochondrial DNA end-binding complex. To confirm that the 68 kDa Ku protein was not a consequence of nuclear protein contamination of mitochondrial preparations, highly purified intact nuclei and mitochondria were treated with proteinase K which traverses the pores of intact nuclei but gains limited access into intact mitochondria. Ku80 in purified intact nuclei was sensitive to treatment with this protease, while the 68 kDa Ku protein characteristic of purified intact mitochondria was resistant. Further, immunocytochemical analysis revealed the co-localization of the N-terminal specific Ku80 monoclonal antibody with a mitochondrial-targeted green fluorescence protein. Mitochondrial localization of the C-terminal Ku80 monoclonal antibody was not observed. These data are consistent with the hypothesis that a C-terminally truncated form of Ku80 is localized in mammalian mitochondria where it functions in a DNA end-binding activity.

Methylation-sensitive amplicon subtraction: a novel method to isolate differentially methylated DNA sequences in complex genomes

A new protocol, termed methylation-sensitive amplicon subtraction (MS-AS), has been developed for the identification and cloning of aberrantly methylated DNA segments in complex genomes. This method is based on the preparation of amplicons from HpaII fragments and combines the normalization of relative DNA sequence abundancy and subtractive hybridization. The normalization step applies a specific form of PCR that permits the exponential amplification of differences in HpaII fragment representations, whereas amplification of common sequences is repressed. In contrast to other subtractive hybridization protocols for genomic DNA analyses, the MS-AS method requires only one cycle of competitive hybridization for the efficient enrichment of target molecules. MS-AS analyses of adenovirus type 12 (Ad12)-transformed or bacteriophage lambda DNA-transgenic hamster cell lines have led to the identification of several CpG-rich cellular gene fragments the methylation of which has been altered in the transgenic as compared to the non-transgenic cell lines. The new method adds to the growing number of genome scanning approaches, including methylation-sensitive representational difference analysis, restriction landmark genomic scanning, and methylation-sensitive arbitrarily primed PCR which all have been used to detect altered methylation sites in cancer cells.

Caspase-dependent apoptosis by ectopic expression of E2F-4.

E2F is a family of transcription factors which regulates cell cycle and apoptosis of mammalian cells. E2F-1-3 localize in the nucleus, and preferentially bind pRb, while E2F-4 and 5 have no nuclear localization signal and preferentially bind p107/p130. E2F-6 suppresses the transcriptional activity of other E2F proteins. DP-1 and 2 are heterodimeric partners of each E2F protein. Using tetracycline-responsive promoters, here we compared the effects of ectopic expression of E2F-1, DP-1 and E2F-4 on cell cycle progression and apoptosis in Chinese hamster cell lines. We found that E2F-4, as well as DP-1 and E2F-1, induced growth arrest and caspase-dependent apoptosis. E2F-4 did not have a marked effect on cell cycle progression, while E2F-1 induced DNA synthesis of resting cells and DP-1 arrested cells in G1. Ectopic expression of E2F-4 did not activate E2F-dependent transcription. Our results suggest that expression of E2F-4 at elevated levels induces growth arrest and apoptosis of mammalian cells through a mechanism distinct from E2F-1 and DP-1.

Defective nuclear localization of p53 protein in a Chinese hamster cell line is associated with the formation of stable cytoplasmic protein multimers in cells with gene amplification.

Many p53 functions require p53 transport into the nucleus. Mutant p53 also generally accumulates in the nucleus of transformed or neoplastic cells. However, examples of cytoplasmic accumulation of wild-type or mutant p53 have also been reported. Various explanations have been provided for defective nuclear localization. Here we propose a novel example of cytoplasmic p53 localization which occurs in cells showing gene amplification and appears to be due to the formation of stable p53 multimers. We studied a methotrexate-resistant Chinese hamster cell line (MTX M) carrying amplified dihydrofolate reductase genes and derived from a cell line with p53 nuclear accumulation. MTX M showed cytoplasmic p53 localization and, on immunoblots, several extra bands in the high molecular weight region, besides the expected 53 kDa band. p53 localization and the appearance of high molecular weight bands appeared to be correlated with the degree of DNA amplification. However, amplification of dihydrofolate reductase itself was not involved. Changing the p53 phosphorylation status quantitatively influenced the formation of high molecular weight bands. Cell fusion experiments demonstrated that p53 cytoplasmic localization in MTX M is a dominant phenotype. This result suggests that the defect causing lack of nuclear localization in this cell line does not reside in the nucleus. In the cytoplasm of MTX M and of wild-type/MTX M heterodikaryons p53 gives rise to protein complexes that are unable to re-enter the nucleus. The formation of such protein complexes is dependent on the amplification of an unknown gene product.

Determinants of NPC1 Expression and Action: Key Promoter Regions, Posttranscriptional Control, and the Importance of a “Cysteine-Rich” Loop

Mutations in the NPC1 gene cause Niemann–Pick type C disease, which is characterized by the accumulation of free cholesterol and other lipids in lysosomes. The NPC1 glycoprotein is located in a late endosomal compartment that transiently interacts with lysosomes. To identify factors regulating NPC1 expression and action, we analyzed the function of the human NPC1 promoter in human-derived ovarian, hepatic, and neuronal cells. A fragment containing the first 208 base pairs upstream from the major transcription initiation site was sufficient to drive near maximal NPC1 promoter activity. Deletion analysis revealed that sequences between base pairs −111 and −37 play an important role in controlling NPC1 transcription. Treatment of proliferating granulosa cells with 30 μM progesterone, which induces a reversible phenocopy of the cholesterol trafficking defect of Niemann–Pick type C disease, increased NPC1 mRNA levels threefold. The protein synthesis inhibitor, cycloheximide, also increased NPC1 mRNA levels, augmenting the progesterone-induced increase in NPC1 mRNA abundance. Progesterone treatment was shown to increase the mRNA half-life, but did not affect NPC1 promoter activity. Cysteine residues in a “cysteine-rich” loop predicted to reside in the intralumenal compartment of vesicles containing NPC1 were mutated, resulting in proteins that were incapable of correcting the cholesterol trafficking defect in CT60 cells, a Chinese hamster cell line in which the endogenous NPC1 gene is inactivated. Converting isoleucine 1061, also predicted to lie within the cysteine-rich loop, to a threonine residue inactivated the protein as well. The I1061T mutation is one of the most common mutations in Niemann–Pick type C disease. All of the cysteine-rich loop mutants were localized to cholesterol-engorged lysosomes in a pattern mimicking the distribution of NPC1 in progesterone-treated cells. A recombinant protein representing the cysteine-rich loop was shown to bind to a zinc–NTA agarose column. We conclude: (1) that cis elements residing in the first 111 base pairs upstream from the transcription start site are critical for transcription of the NPC1 gene; (2) that NPC1 expression is subject to posttranscriptional regulation in response to treatments that disrupt NPC1 function; and (3) that an intralumenal cysteine-rich loop with zinc-binding activity is critical to NPC1's ability to unload lysosomal cargo.