Hamster Cell Mutants Research Articles

A Chinese Hamster Mutant Cell Line with a Defect in the Integral Membrane Protein CII-3 of Complex II of the Mitochondrial Electron Transport Chain

In this study, a respiration-deficient Chinese hamster cell line with a defect in succinate dehydrogenase activity is shown to result from a single base change in a codon in the coding sequence for the membrane anchor protein CII-3 (also referred to as QPs-1). A premature translation stop results in the truncation of 33 amino acids from the C terminus. Bovine cDNA encoding this peptide complements the mutation. There is about 82% identity between these two mammalian proteins. The gene for CII-3 was mapped on human chromosome 1, and because it is also found on minichromosomes characterized by our laboratory, we can localize it on the short arm within 1-2 megabases from the centromere.

Complementation Analysis of the Murine scid Cell Line

It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene.

A novel type of X-ray-sensitive Chinese hamster cell mutant with radioresistant DNA synthesis and hampered DNA double-strand break repair.

It has been shown that the Chinese hamster cell mutant V-C8 is sensitive to different DNA damaging agents, such as mitomycin C (MMC), alkylating agents, UV light, and X-rays. We found that V-C8 is also sensitive to the following radiomimetic agents: bleomycin (approximately 2-fold, based on D10 values), H2O2 (approximately 2-fold), streptonigrin (approximately 11-fold), and etoposide (approximately 8-fold). Two independent spontaneous MMC-resistant revertants isolated from V-C8 cells show a level of cell killing by X-rays, EMS, and UV light which is similar to that of wild-type cells, suggesting that the observed pattern of cross-sensitivity of V-C8 cells to a wide spectrum of DNA damaging agents results from a single mutation. V-C8 cells also display radioresistant DNA synthesis following gamma-irradiation which, however, remained almost unchanged in the V-C8 revertants. The measurement of the level and rate of repair of DNA single- and double-strand breaks (SSBs and DSBs, respectively) by the DNA elution technique showed that the V-C8 mutant has a slower repair of DSBs induced by gamma-rays. The described unique phenotype of V-C8 cells suggested that V-C8 represents a novel type of mutant amongst X-ray-sensitive hamster cell mutants. To confirm this, complementation analysis with other X-ray-sensitive mutants was performed. V-C8 cells were fused with EM9, XR-1, xrs5, sxi-1, V-3, V-E5, irs3, and BLM2 mutant cells, representing different complementation groups. All the obtained hybrids regained X-ray resistance (or bleomycin resistance in the case of V-C8/BLM2 hybrids) similar to that of wild-type cells, indicating that V-C8 represents a new complementation group. The results presented indicate that V-C8 is defective in a gene involved in a pathway operating in the responses to different DNA damaging agents in mammalian cells.

The Effect of Defective DNA Double-Strand Break Repair on Mutations and Chromosome Aberrations in the Chinese Hamster Cell Mutant XR-V15B

The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible changes in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS.

Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation

Murine cells homozygous for the severe combined immune deficiency mutation ( scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PK cs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PK cs, gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PK cs, is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.

Isolation of a Human cDNA That Complements a Mutant Hamster Cell Defective in Methotrexate Uptake

A clone has been isolated from a human lymphoblastic cDNA expression library that complements a mutant Chinese hamster cell defective in the uptake of the folate analogue methotrexate. When transfected with this clone the mutant cells regain the ability to transport the drug and, as a consequence, become sensitive to its cytotoxic action. The clone is 2863 base pairs long and has an open reading frame of 1770 base pairs that codes for a putative protein of 64 kDa. The putative protein has 51 and 50% identity at the amino acid level with the mouse and hamster functions, respectively, involved in the transport of reduced folates. Together these three proteins share 47% identity and have similar predicted structural features. The data are consistent with this human clone encoding either the reduced folate transporter or an auxiliary function that interacts with this transporter.

Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells.

The human DNA repair protein XRCC1 was overexpressed as a histidine-tagged polypeptide (denoted XRCC1-His) in Escherichia coli and purified in milligram quantities by affinity chromatography. XRCC1-His complemented the mutant Chinese hamster ovary cell line EM9 when constitutively expressed from a plasmid or when introduced by electroporation. XRCC1-His directly interacted with human DNA ligase III in vitro to form a complex that was resistant to 2 M NaCl. XRCC1-His interacted equally well with DNA ligase III from Bloom syndrome, HeLa and MRC5 cells, indicating that Bloom syndrome DNA ligase III is normal in this respect. Detection of DNA ligase III on far Western blots by radiolabelled XRCC1-His indicated that the level of the DNA ligase polypeptide was reduced approximately 4-fold in the mutant EM9 and also in EM-C11, a second member of the XRCC1 complementation group. Decreased levels of polypeptide thus account for most of the approximately 6-fold reduced DNA ligase III activity observed previously in EM9. Immunodetection of XRCC1 on Western blots revealed that the level of this polypeptide was also decreased in EM9 and EM-C11 (> 10-fold), indicating that the XRCC1-DNA ligase III complex is much reduced in the two CHO mutants.

Interspecific complementation between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation.

Interspecific and intraspecific hybrids were formed between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation and their radiosensitivities were examined. Chinese hamster cell mutants irs1, irs2 and irs3 and mouse mammary carcinoma cell mutants SX9 and SX10 have been found to belong to five different complementation groups. A radiosensitive mouse lymphoma cell line L5178Y-S has been demonstrated to be different from the X-ray sensitive mouse cell mutants M10 and LX830, both of which are derived from L5178Y cells, in their complementation groups. L5178Y-S is also distinct from SX9 and SX10.

Construction of human chromosome-3-specific radiation hybrids and characterization by Alu-PCR

Using a modification of the procedure developed by Cox et al. [Genomics 4 (1989) 397–407], we isolated and characterized 60 radiation hybrids (RH) prepared by fusing an X-ray-irradiated Chinese hamster-human chromosome 3 (Chr 3) cell line (Q314-2) with a UrdA Chinese hamster mutant cell line. The RH were screened for human DNA content by PCR amplification using primers directed to the human Alu repeat sequences. Over 80% (5060) were scored as positive for the retention of human DNA. Of them, 18 were characterized with Chr-3-specific single-copy DNA probes of known map location. These experiments demonstrated that the RH analyzed contained distinct subrogions of human Chr 3. The RH that we have produced constitute a bank of cellular clones containing small segments of Chr 3. In the accompanying paper [Atchison et al., Gene 151 (1994) 325–328], we present the construction of rare-restrictionsite linking libraries and the sequence tagged site characterization of in situ localized clones.

Somatic cell genetic analysis of two classes of CHO cell mutants expressing opposite phenotypes in sterol-dependent regulation of cholesterol metabolism.

Two different classes of hamster cell mutants (25RA cells and M1 cells) express opposite phenotypes in sterol dependent regulation. In 25RA cells, sterols added in growth medium fail to cause down-regulation of sterol synthesis rate and low density lipoprotein (LDL) receptor activity, while in M1 cells, removal of lipids from growth medium fail to cause up-regulation of sterol synthesis rate and LDL receptor activity. Cell hybridization analysis showed that the 25RA phenotype is semidominant, while the M1 phenotype is recessive. Using 25RA as the parental cells, we isolated eight independent mutant cells (DM cells) and showed that all of them belong to the same genetic complementation group as the M1 mutant, indicating that the normal (unmutated) M1 gene product(s) is required to express the 25RA phenotype. We next performed gene transfer experiments using hamster cell genomic DNAs containing the functional human M1 gene as the donor, and the double mutant cell DM7 as the recipient. The resultant transfectant cells express the 25RA cell phenotype (instead of the wild-type cell phenotype). This result, along with the results obtained from cell hybridization analysis, shows that the 25RA and M1 cell phenotypes are caused by mutations at two different genes.

Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks.

The Ku autoantigen is a well-characterized heterodimer of 70 and 86 kDa that binds to DNA ends, but its cellular function has been obscure. An electrophoretic mobility-shift assay and Ku antisera were used to show that Ku or a closely related protein was deficient in three mutant hamster cell lines from x-ray-sensitive complementation group 5, which is characterized by defects in DNA double-strand break repair and V(D)J recombination. Furthermore, Ku protein expression was restored when the cells reverted to x-ray resistance. The Ku p86 gene maps to human chromosome 2q33-35, and group 5 cells are rescued by almost precisely the same region, 2q34-36. Thus, biochemical and genetic evidence suggests that Ku is involved in pathways for DNA recombination and repair. By its association with a DNA-dependent protein kinase activated by DNA ends, Ku may also initiate a signaling pathway induced by DNA damage, perhaps for cell cycle arrest.

Molecular cloning of the human nucleotide-excision-repair gene ERCC4.

ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a secondary transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.

Low hprt mRNA levels and multiple hprt mRNA species in 6-thioguanine-resistant Chinese hamster cell mutants possessing nonsense mutations

Previous studies suggested that many Chinese hamster ovary (CHO) cell hprt mutants having point mutations in the protein coding region also have low steady-state hprt mRNA concentrations. In addition, polymerase-chain reaction (PCR) amplification of hprt cDNA synthesized from some of these mutants results in multiple products containing deleted exons indicating that these mutants posses multiple species of hprt mRNA. In this study, we have used northern blot analysis to quantify the concentrations of hprt mRNA in 86 mutants known to possess point mutations leading to either missense or nonsense mutations. 28 of 35 nonsense mutants (80%), but only 7 of 51 missense mutants (14%), had < 50% of the hprt mRNA concentration found in parental CHO cells. Furthermore, all the nonsense mutants with premature termination codons in the internal exons of the gene ( i.e., exons 3, 4, 5, 6 and 7) showed a significant reduction (averages < 16% of parental) in the steady-state levels of hprt mRNA, while nonsense mutants with termination codons situated in the extreme 5′ and 3′ regions of the gene had near parental hprt mRNA levels. In the same collection of mutants, the proportion of mutants producing multiple cDNA PCR products was much greater ( 18 35 ) for mutants having nonsense mutations than for mutants with missense mutations ( 2 51 ). All nonsense mutants with mutations in exons 3, 4 and 5 produced multiple species, while all those with mutations in exons 7, 8 and 9 produced a single PCR product. These results suggest that sequence changes in mammalian genes that affect protein chain length can also affect mRNA concentration and the splicing of pre-mRNA molecules.

Mutagenic response and repair of cis-DDP-induced DNA cross-links in the Chinese hamster V79 cell mutant V-H4 which is homologous to Fanconi anemia (group A)

Previously, it has been shown that the V-H4 mutant of Chinese hamster V79 cells is homologous to Fanconi anemia (FA) group A cells. This hamster cell mutant shows a specific sensitivity to DNA cross-linking agents; therefore, the induction and repair of DNA cross-links were studied in V-H4 and wild-type V79 cells after cis-DDP treatment by the DNA alkaline elution technique. A significant difference in repair of these lesions in V-H4 and wild-type cells was observed. After the cis-DDP treatment (24 h) about 3 times more cross-links remained in V-H4 cells in comparison to the parental V79 cells. These results indicate that the process of cross-link repair in V-H4 cells is hamperedwhen compared to that of wild-type cells. To assess the effect of slower removal of DNA cross-links on the mutability of V-H4, the induction of mutants at the hypoxanthine-guanine phosphoribosyltransferase locus (HPRT) by cis-DDP was studied in V-H4 and V79 cells. Despite the increased cytotoxicity of cis-DDP to V-H4 cells, the mutation induction at the HPRT locus was not significantly different in both cell lines, but when the frequency of the hprt mutants was plotted against survival, hypomutability was observed in V-H4 cells after the cis-DDP treatment.

Functional complementation studies with X-ray-sensitive mutants of Chinese hamster cells closely resembling ataxia-telangiectasia cells

In order to isolate a human gene complementing the defect in A-T-like hamster cell mutants, the mutants were used as recipients for genomic DNA transfection, using either HeLa chromosomal DNA or DNA from a human cosmid library. Three primary transformants with an intermediate X-ray sensitivity and almost normal sensitivity to MMS, but retaining radioresistant DNA synthesis (RDS), were obtained. To identify the human chromosome that complements the defect in the A-T-like mutants, and to assess the degree of complementation for survival and RDS, microcell-mediated chromosome transfer was used. At least 20 independent hybrid clones between the mutant and each one of the human chromosomes 1, 2, 4, 5, 15, 17 or 18 were isolated. All hybrid clones remained X-ray sensitive, except one with chromosome 4, and another with chromosome 15, both showing an intermediate X-ray sensitivity. By using in situ hybridization we found that this partial correction was due to the presence of a mouse chromosome. In these two hybrids containing the mouse chromosome together with human chromosome 4 or 15, RDS was fully complemented only in the hybrid with chromosome 4 but not in the one containing chromosome 15, suggesting that RDS and X-ray sensitivity may be complemented independently.

Genetic analysis of mitomycin C-hypersensitive Chinese hamster cell mutants.

The genetic diversity of Chinese hamster cell mutants exhibiting hypersensitivity to the bifunctional alkylating agent mitomycin C has been examined. The eight mutants irs3, VH4, UV1, MC5, MMC1, MMC3, MMC4 and MMS2, are between 4- and 30-fold more sensitive to mitomycin C than their respective wildtype parental lines. A number of the mutants show phenotypic similarities to cultured cells from the human cancer-prone syndrome Fanconi's anaemia. Hybrids were formed between pairs of mutants using the thioguanine/ouabain resistant (TOR) hybridization and hypoxanthine/aminopterin/thymidine (HAT)/ouabain selection system and the mitomycin C response of pooled populations of hybrids assessed by constructing survival curves. In every case, hybrids formed between pairs of mutants exhibited a mitomycin C response indistinguishable from that of wildtype cells, indicating complementation. Therefore, the eight mutant lines examined represent eight distinct complementation groups for mitomycin C-hypersensitivity. The results are in contrast to the complementation analysis of UV-sensitive Chinese hamster cell mutants and indicate that the response of mammalian cells to mitomycin C-induced DNA damage is complex and involves a large number of genes.

Nuclear scaffold organization in the X-ray sensitive Chinese hamster mutant cell line, xrs-5.

Nuclear organization was probed in the radiation-sensitive Chinese hamster ovary CHO) cell line, xrs-5, and compared with parental CHO K1 cells using the resinless section technique and DNase I digestions. The resinless section data showed no gross morphological differences in core filaments from the nuclear scaffolds of unirradiated CHO K1 and xrs-5 cells. However, the nuclear scaffolds of irradiated xrs-5 cells (1 Gy) had significantly increased ground substance. Irradiated and unirradiated CHO K1 cell nuclear scaffolds were morphologically identical. These data suggest that both CHO K1 and xrs-5 cell nuclear scaffolds had internal nuclear scaffolding networks that could provide DNA attachment sites. The rate of DNase I digestion of unirradiated CHO K1 and xrs-5 was not significantly different, but the extent of digestion was greater in unirradiated CHO K1 cells that in xrs-5 cells suggesting that less xrs-5 cell chromatin at DNA attachment points is accessible to the enzyme DNase I. The extents of digestion in irradiated (1 Gy) CHO K1 and xrs-5 cell nuclei also differed but the relationship was reversed. The irradiated xrs-5 cell samples were digested to a greater extent compared with CHO K1 cells. These chromatin digestion data suggest that the matrix attachment regions in xrs-5 cells are different from CHO K1 cells. The different DNA attachment organization in the xrs-5 cells may play a role in modulating radiation sensitivity.

Cellular characteristics of Chinese hamster cell mutants resembling ataxia telangiectasia cells

The radiosensitive Chinese hamster V79 cell mutants (V-C4, V-E5 and V-G8), isolated previously in our laboratory, have been shown to resemble human ataxia telangiectasia (A-T) cells. These hamster cell mutants were further characterized with respect to cross-sensitivity to different radiomimetic agents and to mutation induction by X-rays. The data on cell survival (D10 Values) show that they are hypersensitive to adriamycin (2–3-fold increase), etoposide (3-fold for V-G8 and 6-fold for V-E5 and V-C4), calicheamicin γ1I (4-fold) and streptonigrin (3-fold for V-G8 and V-C4, and 12-fold for V-E5). The frequency of X-ray-induced hprt mutations is slightly enhanced in the hamster mutant cells treated with the same dose. However, the mutants show similar mutability as parental V79 cells when considering the same survival level. The overall conclusion from these studies is that these hamsters cell mutants mimic the phenotypic characteristics observed in cultured cells from A-T patients and therefore, may be defective in the same repair pathway as their human counterparts.

Loss of Transcriptional Activation of Three Sterol-Regulated Genes in Mutant Hamster Cells

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.

Loss of transcriptional activation of three sterol-regulated genes in mutant hamster cells.

Cholesterol biosynthesis and uptake are controlled by a classic end product-feedback mechanism whereby elevated cellular sterol levels suppress transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low-density lipoprotein receptor. The 5'-flanking region of each gene contains a common cis-acting element, designated the sterol regulatory element (SRE), that is required for transcriptional regulation. In this report, we describe mutant Chinese hamster ovary (CHO) cell lines that lack SRE-dependent transcription. Mutant cell lines were isolated on the basis of their ability to survive treatment with amphotericin B, a polyene antibiotic that kills cells by interacting with cholesterol in the plasma membrane. Four mutant lines (SRD-6A, -B, -C, and -D) were found to be cholesterol auxotrophs and demonstrated constitutively low levels of mRNA for all three sterol-regulated genes even under conditions of sterol deprivation. The mutant cell lines were found to be genetically recessive, and all four lines belonged to the same complementation group. When transfected with a plasmid containing a sterol-regulated promoter fused to a bacterial reporter gene, SRD-6B cells demonstrated constitutively low levels of transcription, in contrast to wild-type CHO cells, which increased transcription under conditions of sterol deprivation. Mutation of the SREs in this plasmid prior to transfection reduced the level of expression in wild-type CHO cells deprived of sterols to the level of expression found in SRD-6B cells. The defect in SRD-6 cells is limited to transcriptional regulation, since posttranscriptional mechanisms of sterol-mediated regulation were intact: the cells retained the ability to posttranscriptionally suppress HMG-CoA reductase activity and to stimulate acyl-CoA:cholesterol acyltransferase activity. These results suggest that SRD-6 cells lack a factor required for SRE-dependent transcriptional activation. We contrast these cells with a previously isolated oxysterol-resistant cell line (SRD-2) that lacks a factor required for SRE-dependent transcriptional suppression and propose a model for the role of these genetically defined factors in sterol-mediated transcriptional regulation.