Expression of the ATDC (ataxia telangiectasia group D-complementing) gene in A431 human squamous carcinoma cells.

The ATDC gene was originally identified by its ability to complement the radiosensitivity defect of an ataxia telangiectasia (AT) fibroblast cell line. Because hypersensitivity to ionizing radiation is an important feature of the AT phenotype, we reasoned that ATDC may function generally in the suppression of radiosensitivity. Previous work in our laboratory focused on radiosensitization mechanisms in human squamous carcinoma (SC) cells, especially A431 cells. To establish a basis for investigating the role of ATDC in radiation-responsive signaling pathways in human SC cells, we characterized ATDC message and protein expressions in A431 cells. ATDC message expression was also compared among human epidermoid cells (A431 cells, HaCaT spontaneously immortalized human keratinocytes and normal human epidermal keratinocytes) and a normal human fibroblast cell line (LM217). We made the following major observations: (i) the relative abundance of ATDC message is substantially higher in the epidermoid cells than in the fibroblast cell line, which has a message level comparable to those reported for other fibroblast lines; (ii) ATDC is constitutively phosphorylated on serine/threonine in A431 cells; (iii) in A431 cells, ATDC is a substrate for the serine/threonine protein kinase C (PKC) but not the epidermal growth factor (EGF) receptor tyrosine kinase; and (iv) EGF decreases ATDC message and protein expressions in A431 cells after a 24-hr exposure. The phosphorylation studies suggest that the ability of ATDC to modulate cellular radiosensitivity may be mediated in part through a PKC signaling pathway.

Expression of a candidate ataxia-telangiectasia group D gene in cultured fibroblast cell lines and human tissues.

A candidate gene for ataxia-telangiectasia group D (ATDC) has been cloned (Kapp et al. 1992), sequenced and found to be a member of a recently reported gene family (Leonhardt et al. 1994). Transcriptional behaviour of ATDC has been examined in a number of cell lines and human tissues using a 3.0 kb cloned cDNA as a probe. Three normal and two ataxia-telangiectasia (A-T) group D, non-transformed fibroblast cell lines produced a 2.4 kb mRNA transcript. The size of mRNA transcripts seen and the level of expression differ in different human tissues. Many tissues have multiple transcripts of ATDC and the most prominent transcripts observed were 3.0, 2.4 and 1.6 kb. Two out of three SV-40 transformed normal and one SV-40-transformed A-T group D fibroblast cell lines demonstrated no transcription of ATDC by RNA blotting analysis. These results suggest that SV-40 transformation may affect the expression of ATDC. Reverse transcription-polymerase chain reaction analysis showed that ATDC was expressed at low levels in all of these cells. Additional Northern blot analysis demonstrated that X-irradiation with 10 Gy had no effect on ATDC expression at 1, 4 and 24 h after irradiation in either SV-40-transformed normal or in SV-40-transformed A-T group D fibroblast cell lines. Further understanding of ATDC will require cloning of additional transcripts and studies of ATDC protein behaviour.

Nucleotide sequence analysis of a candidate gene for ataxia-telangiectasia group D (ATDC).

A radioresistant cell clone (1B3) was previously isolated after transfection of an ataxia-telangiectasia (AT) group D cell line with a human cosmid library. A cosmid rescued from the integration site in 1B3 contained human DNA from chromosome position 11q23, the same region shown by both genetic linkage and chromosome transfer to contain the genes for AT complementation groups A/B, C, and D. A gene within the cosmid (ATDC) was found to produce mRNAs of different sizes. A cDNA for one of the most abundant mRNAs (3.0 kb) was isolated from a HeLa cell library. In the present study, we sequenced the 3.0-kb cDNA and the surrounding intron DNA in the cosmids. We used polymerase chain reaction, with primers in the introns, to confirm the number of exons and to analyze DNA from AT group D cells for mutations within this gene. Although no mutations were found, we do not rule out the possibility that mutations may be present within the regulatory sequences or coding sequences found in other mRNAs specific for this gene. From the sequence analysis, we found that the ATDC gene product is one of a group of proteins that share multiple zinc finger motifs and an adjacent leucine zipper motif. These proteins have been proposed to form homo- or heterodimers involved in nucleic acid binding, consistent with the fact that many of these proteins appear to be transcriptional regulatory factors involved in carcinogenesis and/or differentiation. The likelihood that the ATDC gene product is involved in transcriptional regulation could explain the pleiomorphic characteristics of AT, including abnormal cell cycle regulation.

A critical look at the association of human genetic syndromes with sensitivity to ionizing radiation.

Individuals with inherited heterozygosity for mutations involving tumor suppressor genes may be at greater risk for ionizing radiation-induced cancer even though their cells may not show increased cytotoxicity or chromosome damage. In addition, many human genetic syndromes have been reported to show increased sensitivity to ionizing radiation. In most instances the effects are small and/or not reproducible. Only in the genetic disease ataxia-telangiectasia (AT) is radiosensitivity consistently outside the normal range. Even AT heterozygotes (0.68 to 7.7% of the population) appear to be slightly radiosensitive, and their elevated cancer risk may result from exposure to ionizing radiation. Current research has concentrated on the isolation of the gene or genes responsible for this disease.

Critical steps for induction of chromosomal aberrations in CHO cells heated in S phase.

The following four effects on DNA replication are observed in cells heated in S phase of the cell cycle: (1) inhibition of replicon initiation, (2) delay in DNA chain elongation into multicluster-sized molecules > 160S, (3) reduction in fork displacement rate, and (4) increase in single-stranded regions in replicating DNA. Since cells heated in S phase manifest chromosomal aberrations when they enter metaphase, whereas cells heated in G1 do not, we attempted to determine if the effects on DNA replication are critical for the induction of chromosomal aberrations by studying these same effects during DNA replication when synchronous CHO cells had been heated (10 min at 45.5 degrees C) in G1 phase. Following a heat-induced G1 block (12 h), we found previously that when the cells entered S phase, replicon initiation was functional and chain elongation into multicluster-sized molecules > 160S was delayed but completed during S phase. In the present study, we find that the fork displacement rate was near normal and that there was no increase in single-stranded DNA. Additionally, an increase in excess nuclear protein induced in the heated G1-phase cells returns to a normal level by about 12 h, just prior to when the cells enter S phase. Since the excess nuclear protein remains for many hours in heated S-phase cells, we hypothesize that the excess nuclear protein is responsible for the drastic reduction in the fork displacement rate and the associated increase in single-stranded DNA. Furthermore, we hypothesize that this persistent increase in single-stranded DNA during replication is a critical step for the induction of chromosomal aberrations in heated S-phase cells. Consistent with this hypothesis, we observed that aphidicolin (1-2 micrograms/ml) treatment of S-phase cells for 13-16 h, which results in a twofold increase in single-stranded DNA during the inhibition of DNA synthesis, also induces chromosomal aberrations. Possibly, endogenous endonucleolytic attack occurs opposite these sites of single-stranded DNA, thus creating double-strand breaks which either can remain unrepaired or are misrepaired to account for the chromatid breaks and exchanges, respectively, observed as cells complete their cell cycle and enter metaphase.

Cloning of a candidate gene for ataxia-telangiectasia group D.

Transfection, with a human cosmid clone library, of an ataxia-telangiectasia (AT) cell line (AT5BIVA) from complementation group D previously resulted in the isolation of a cell line (1B3) with partially restored resistance to ionizing radiation. We rescued the integrated cosmid sequences within 1B3 and obtained two cosmid clones that contained overlapping DNA from chromosomal region 11q23, previously shown to be the region containing the AT gene(s) from three complementation groups. Isolation of an apparently full-length 3.0-kb cDNA from a HeLa cell library demonstrated a previously unidentified gene (ATDC) within these cosmid clones. The transfected copy of the ATDC gene in 1B3 is truncated at the 3' end but is a complete transcription unit, because of the presence of SV40 termination sequences within the adjacent cosmid DNA. After further screening of cosmid clones from a chromosome 11 library, we identified contiguous DNA that contained the missing portion of the gene. Southern blot analysis indicated that the ATDC gene is present in a single copy in the human genome; however, RNA blot analysis revealed mRNA of several sizes (1.8, 2.6, 3.0, 4.7, and 5.7 kb) that varied among different cell lines. Because no large rearrangements were detected in AT5BIVA cells by Southern or RNA blot analysis, any alteration in the ATDC gene in this cell line would involve a point mutation or a small rearrangement. Transfection of the AT5BIVA cell line with one of the cosmids partially restored radioresistance. Analysis of 100 X-radiation hybrid cell lines containing various fragments from the chromosomal region 11q23 showed that the ATDC gene is closely linked to THY1. The ATDC gene therefore lies outside the linkage region predicted to contain the AT gene(s) for complementation groups A and C, indicating a separate locus for the AT complementation group D gene.

Replication intermediates as substrates for DNA rearrangements.

A model for the formation of DNA rearrangements in somatic cells is presented. Two double-strand breaks at the junctions between unreplicated DNA and newly replicated DNA generate four double-stranded DNA molecules that can recombine to form tandem duplications, inversions, deletions and extrachromosomal DNA circles.

Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells.

SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid that contains a normal human DNA library and a selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. After a three-part selection protocol for G418 resistance and radioresistance, a cell line stably resistant to ionizing radiation was recovered. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one that was stably resistant to radiation. Resistance to ionizing radiation of both the primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G2 chromatid aberrations; both cell lines retained AT-like radioresistant DNA synthesis. These results suggest that, because radioresistance in the transfected cells was not as great as that in normal human cells, the two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene.

DNA fork displacement rate measurements in heated Chinese hamster ovary cells.

DNA fork displacement rates (FDR) were measured in Chinese hamster ovary (CHO) cells heated at either 43.5 degrees C or 45.5 degrees C for various times. The inhibition of fork movement rate by heat was both time and temperature dependent, i.e., 10-20 min at 43.5 degrees C or 5 min at 45.5 degrees C was required to decrease the FDR to 20-30% of the control rate of 1 micron/min. Following heating, the reduced FDR was found to be constant for at least 75 min. The observed effects of heat on reduced rates of DNA replicon initiation and chain elongation and the increase in DNA with single-stranded regions could be explained by the heat sensitivity of the FDR. Any of these alterations in the DNA replication process may lead to many opportunities for abnormal DNA and/or protein interactions to occur which ultimately may lead to the observed formation of chromosomal aberrations.

Absence of DNA overreplication in Chinese hamster cells incubated with inhibitors of DNA synthesis.

Previous reports have suggested that transient inhibition of DNA synthesis by chemicals or ultraviolet light causes some of the DNA to replicate more than once in one cell cycle, i.e., that it induces overreplication of DNA. The data that led to this suggestion were obtained from cesium chloride equilibrium density gradient analyses, in which cells were incubated with bromodeoxyuridine so that the DNA synthesized after incubation with the inhibitor could be densitometrically separated from the DNA that had been radioactively labeled before incubation with the inhibitor. An unresolved problem with these analyses was that the data also suggested that overreplication must have occurred in control cells, i.e., those not incubated with an inhibitor of DNA synthesis. We show here that the latter result is probably due to an artifact of cesium chloride equilibrium density gradient analysis, probably because of nonspecific trapping of DNA in regions of the gradients where there are large amounts of DNA. We also used another protocol that avoids this artifact; with this protocol any overreplicated DNA would be found where heavy-heavy DNA bands and nonspecific trapping cannot occur. When this protocol was used there was no evidence that transient inhibition of DNA synthesis induces overreplication of DNA.

Establishment and characterization of two human cell lines with amplified dihydrofolate reductase genes.

Two SV40-transformed human cell lines, GM637, derived from a normal human subject, and GM5849, derived from a patient with ataxia-telangiectasia (A-T), were grown in increasing concentrations of the cytotoxic agent methotrexate (MTX). The GM637 line was naturally more resistant to methotrexate than was GM5849 and, over a 5-month period, became resistant even to very high concentrations (up to 100 microM). The GM5849 line became resistant to 500 nM methotrexate during the same period. However, dot blot and Southern blot analyses showed that both cell lines had amplified their dihydrofolate reductase (dhfr) genes to about the same extent, approx. 50-fold. Using the GM5849 line with amplified dhfr, we attempted to determine if interruption of DNA synthesis by hydroxyurea would cause DNA to be replicated twice within a single cell cycle, as has been reported for Chinese hamster ovary cells. No evidence for such a phenomenon was obtained.

Spontaneous and 3-aminobenzamide-induced sister-chromatid exchange frequencies estimated by ring chromosome analysis.

Ring chromosomes offer an opportunity to measure sister-chromatid exchange (SCE) frequencies without the use of an agent to differentiate sister chromatids: SCE frequencies can be determined from the number of dicentric rings formed in cells from a cell line carrying a monocentric ring chromosome. Ash is a pseudotetraploid Chinese hamster ovary cell line in which approximately 40% of metaphase cells have a large ring chromosome. We have used this cell line to investigate the spontaneous rate of SCE by determining the rate of dicentric ring formation and have compared this with the rate of loss of the ring chromosomes over time. In the absence of both [3H]thymidine and bromodeoxyuridine, the spontaneous rate of SCE in Ash cells was 0.12 SCEs/ring/cell cycle; this rate was increased by bromodeoxyuridine, by the polyfunctional alkylating agent mitomycin C, and by the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. This indicates that spontaneous SCE occurs in this line and that not all 3-amino-benzamide-induced SCEs are dependent upon incorporated bromodeoxyuridine. Ring chromosomes were not lost over time as rapidly as predicted by the SCE frequencies observed. Non-disjunction of the dicentric ring, or anaphase bridge breakage followed by reunion to form one or two monocentric rings, are the most likely explanations for this discrepancy.

Chromosomal changes without DNA overproduction in hydroxyurea-treated mammalian cells: implications for gene amplification.

It has been reported that a 6-h incubation of early S-phase Chinese hamster cells with hydroxyurea promotes DNA overproduction, i.e., replication of DNA a second time within a single cell cycle, and that this could be the basis for gene amplification in drug-treated mammalian cells. When we incubated methotrexate-resistant Chinese hamster cells that were approximately 2 h into the S phase with hydroxyurea for 6 h, DNA that had been replicated before the incubation with hydroxyurea (early S-phase DNA) was replicated again within 11 h after the hydroxyurea treatment. However, incubation with colchicine or Colcemid after hydroxyurea treatment virtually abolished this overreplication, as well as that of the amplified dihydrofolate reductase genes in these cells, indicating that the second replication had occurred in a second cell cycle. Cells collected in the first mitosis after incubation with hydroxyurea never contained overreplicated DNA but did contain abundant chromosome aberrations. Early S-phase DNA replicated again on schedule during the first few hours after mitosis. Asymmetric segregation of chromosome fragments or unequal sister chromatid exchange may be the actual basis for gene amplification in drug-treated mammalian cells.

A cytogenetic investigation of DNA rereplication after hydroxyurea treatment: implications for gene amplification.

Although the mechanisms leading to gene amplification are poorly understood, it has recently been proposed that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome within a single cell cycle. We sought evidence for rereplication of DNA as a basis for gene amplification through two cytogenetic techniques: differential staining for sister-chromatid exchange analysis and premature chromosome condensation. Synchronized Chinese hamster ovary cells were incubated continuously with bromodeoxyuridine and treated with hydroxyurea (HU) when cells were approximately 2 h into the S phase. After 6 h exposure to HU, the drug was removed and at 3 h intervals thereafter metaphase cells were collected and the chromosomes were stained by the fluorescence-plus-Giemsa procedure. No staining patterns consistent with rereplication of DNA were observed. Since HU causes cytogenetic damage, the premature chromosome condensation technique was used to determine the kinetics of chromosome damage after removal of HU. Extensive G2 chromosome damage within 1 h after removal of HU from the medium was found, although cesium chloride gradient analysis showed that there was no rereplication of DNA during this time. Contrary to a previous report, these results provide no evidence that incubation of cells with HU during S phase induces rereplication of DNA within a single cell cycle. The results observed are consistent with the hypothesis that drug-induced aberrations and the subsequent abnormal segregation of chromosomal fragments are the first steps in the process that leads to gene amplification in drug-treated mammalian cells.

DNA-fork displacement rates in cultured mammalian cells with mutations in ribonucleotide reductase.

DNA-replication fork displacement rates were measured in mouse S49 lymphosarcoma cell lines and in derivatives of those cell lines. One of the derivatives lacks dCMP deaminase activity and two others bear defined mutations in ribonucleotide reductase. We also examined a revertant cell line that was selected from one of the ribonucleotide reductase mutants and has regained normal ribonucleotide reductase activity. Our results show a correlation between decreased fork-displacement rates and alterations in ribonucleotide reductase, suggesting a possible involvement of this enzyme in the replication apparatus.

Replicon size and excision repair as factors in the inhibition and recovery of DNA synthesis from ultraviolet damage.

Initiation of DNA replication and chain growth, analyzed by alkaline sucrose gradient sedimentation, was interrupted to different extents in different cell types by irradiation with ultraviolet light. Within the first hour of irradiation DNA replication was reduced in a manner that depended on the average number of lesions per replicating unit (replicon). At low numbers of lesions per replicon, inhibition of replicon initiation was the predominant response; at higher numbers of lesions per replicon, blockage of chain growth was also observed. After irradiation with a dose that initially blocked chain growth, the rate at which cells recovered their ability to synthesize increasingly more and larger size DNA was a function both of replicon size and of excision repair capacity. Cells with small replicons recovered more rapidly than cells with large replicons, and excision repair-deficient cells recovered less rapidly than excision-competent cells. These observations indicate that excision repair capacity and replicon size play major roles in the response of DNA replication to ultraviolet damage.

Effects of 3-aminobenzamide on DNA synthesis and cell cycle progression in Chinese hamster ovary cells.

3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, is a potent inducer of sister chromatid exchanges (SCEs). Because of the possible relation between SCEs and DNA synthesis, the effects of 3AB on DNA synthesis and cell cycle progression in Chinese hamster ovary (CHO) cells were examined. Unlike all other SCE-inducing agents whose effects on DNA synthesis have been studied, short term exposures (30-120 min) of 3AB did not inhibit the overall rate of DNA synthesis and this result was independent of the amount of bromodeoxyuridine (BrdU) in the DNA. Longer exposure times (greater than 24 h) did result in an extended S phase, but this was not due to an effect on the rate of DNA chain elongation. 3AB also delayed the entry of cells into S phase. The overall cell cycle delay was dose dependent, approaching 9 h after a 54 h exposure to 10 mM 3AB. Earlier reports that 3AB is neither mutagenic nor cytotoxic were confirmed. Thus 3AB acts to increase SCE frequency by a mechanism distinct from that which causes cytotoxicity and mutagenicity, and does not involve any inhibition in the rate of DNA chain growth.