Dennis Hellgren

Expression of the human RAD51 gene during the cell cycle in primary human peripheral blood lymphocytes

The S. cerevisiae RAD51 gene product exerts important functions in meiotic and mitotic recombination, as well as in the repair of DNA double-strand breaks. We have studied the expression of the human RAD51 (HsRAD51) gene in primary human peripheral blood lymphocytes (PBLs). The HsRAD51 mRNA level increased three fold in mitogen stimulated PBLs, with a peak in the late S phase. A five fold increase of HsRAD51 protein levels was observed in late G2. Specific inhibition of DNA synthesis with aphidicolin did not block the induction of the HsRAD51 protein, indicating that HsRAD51 expression is independent of DNA replication. In contrast, after inhibition of RNA synthesis with actinomycin D and protein synthesis with cycloheximide, the HsRAD51 protein level decreased rapidly. Taken together, these results indicate that the HsRAD51 gene is transcriptionally regulated in human PBLs, and exerts its function during the late S and G2 phases of the cell cycle.

Duplication in the hypoxanthine phosphoribosyl-transferase gene caused by Alu-Alu recombination in a patient with Lesch Nyhan syndrome

We have determined the structure, at the nucleotide sequence level, of a duplication in the hprt gene in a patient with Lesch-Nyhan syndrome (LN). The duplication extends over exons 7 and 8 and approximately 1.8 kb of the surrounding hprt sequence. The duplication junction is localized within two Alu sequences and has apparently been generated by unequal homologous recombination. This is the second reported case of a partial duplication of the hprt gene in an LN patient, and the first that involves an Alu-Alu recombination.

Proteolytic cleavage of HsRad51 during apoptosis

The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T‐lymphocyte cell lines, Jurkat and PFI‐285. Apoptosis was induced by camptothecin or anti‐Fas monoclonal antibody (anti‐Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP‐ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S‐100 extract in a cell‐free system. In each case, cleavage of HsRad51 was abolished by acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde (Ac‐DEVD‐CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase‐2, ‐3 or ‐6 to ‐10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA‐dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.

Mutagen-induced recombination in mammalian cells in vitro

It is now clear from in vitro studies that mutagens induce recombination in the cell, both homologous and nonhomologous exchanges. The recombination events induced are extrachromosomal events, exchanges between extrachromosomal DNA and chromosomes, and inter- as well as intrachromosomal exchanges. However, not all types of DNA damage can induce recombination. The mechanisms involved in the induction process are not known but may involve activation of DNA repair systems. In addition, stimulation of mRNA transcription by mutagens, different recombination pathways and how the assay system is constructed may affect the frequency and characteristics of the observed recombination events.

Different SCE-inducing effects of HN2 and MMS in early and late G1 in human lymphocytes

The induction of SCE was studied in PHA-stimulated human lymphocytes exposed to nitrogen mustard (HN2) or methyl methanesulfonate (MMS) for various time periods in the G1 phase. HN2 was found to induce about 10 times more SCE when cells were exposed in late G1 (24 h after PHA) as compared to early G1 (immediately after PHA). In contrast, only a small difference was observed between cells exposed to MMS in late or early G1. The results suggest that different types of SCE-inducing alkylating damage agents are removed at widely different rates in human G1-lymphocytes.

Effects of arabinosyl-cytosine on thymidine triphosphate pools and polyoma DNA replication

Abstract Arabinosyl cytosine at very low concentrations (5–100 nmolar) inhibits the incorporation of [ 3 H]thymidine into polyoma DNA of infected mouse fibroblasts without affecting the labeling of the [ 3 H]dTTP pool. The specific activities of these pools were determined by a new simple method. Inhibition of DNA synthesis affects chain elongation and not initiation of new rounds of replication.

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells.

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.

The frequency of illegitimate TCRβ/γ gene recombination in human lymphocytes: influence of age, environmental exposure and cytostatic treatment, and correlation with frequencies of t(14;18) and hprt mutation

Chromosome translocations in lymphoid malignancies often involve V(D)J recombinase mediated events giving rise to aberrant T-cell receptor (TCR) and immunoglobulin genes, which have been suggested to be useful as markers of genomic instability, genotoxic exposure and cancer risk. Illegitimate rearrangements involving the TCRbeta/gamma loci on chromosome 7 create TCRbeta/gamma hybrid genes which occur at low frequency in peripheral blood lymphocytes (PBLs) of normal healthy individuals. To evaluate the utility of this marker, we studied the possible effects of age and genotoxic exposures on the TCRbeta/gamma gene variant frequency (VF), and compared the frequencies of hypoxanthine guanine phosphoribosyl transferase (hprt) mutation, hprt exon 2/3 deletion, t(14;18) and TCRbeta/gamma gene rearrangements in cells from the same donors. The TCRbeta/gamma VF ranged five-fold among 16 middle aged blood donors with a mean of 0.74+/-0.29/10(5) PBLs, which is consistent with our previous estimate in healthy subjects. The TCRbeta/gamma VF was found to increase from birth until early adult life, and then to decrease with increasing age. Four testis cancer patients, who 6 years earlier had been treated with etoposide and other cytostatic drugs, showed TCRbeta/gamma VF similar to that in healthy controls. No increase of the TCRbeta/gamma VF was found among non-smoking PAH-exposed aluminum smelter workers compared to non-smoking controls. Smoking smelter workers showed decreased TCRbeta/gamma VF compared to non-smoking workers and controls, but in a follow-up study 2 years later the difference was no longer statistically significant, although the smoking smelter workers still showed a lower TCRbeta/gamma VF than the controls. No correlation was obtained between the TCRbeta/gamma VF and the t(14;18) or hprt mutant frequency (MF) in a group of healthy individuals. However, there was a statistically significant correlation between the TCRbeta/gamma VF and the hprt exon 2/3 deletion frequency in PBL DNA from the same donors. These results show that the TCRbeta/gamma VF in healthy individuals changes with age and correlates with the frequency of hprt exon 2/3 deletion, another marker of aberrant V(D)J recombination in T-cells. However, no effect of smoking or present or previous exposure to genotoxic agents on TCRbeta/gamma VF was observed in this study. Thus, further studies are needed to prove the utility of TCRbeta/gamma gene rearrangement as a marker of genotoxic exposure.

Unequal SCE is a rare event in homologous recombination between duplicated neo gene fragments in CHO cells

The frequency of sister-chromatid exchange (SCE) was studied in Chinese hamster ovary (CHO) cell lines with stable insertions of the vector pIII-14gpt which contains 2 truncated neomycin resistance (neo) gene fragments. Recombination between regions of homology in the 2 fragments can restore a functional neo gene and make the cell resistant to the antibiotic G418, a neomycin analogue. Unequal SCE would be one of several possible mechanisms for this event. The observed spontaneous rate of formation of G418-resistant subclones was approximately 6.4 x 10(-6) per cell per generation, as compared to the estimated spontaneous frequency of 3 SCE per cell per generation. Given this SCE frequency, the probability of an SCE occurring in a target site of about 1600 bp (the distance separating the homologous regions in the neo fragments) would be about 8 x 10(-7) per cell per generation, or approximately one tenth of the estimated rate of recombination. Treatment of the cells with methyl methanesulfonate (MMS, 50 x 10(-6) M) induced about 80-90 SCE per cell, corresponding to a probability of 2 x 10(-5) SCE per 1600-bp target per cell. In the same cell culture, MMS treatment induced 4-8 x 10(-4) recombination events per cell giving rise to G418 resistance. Cells treated with HN2 (up to 4 x 10(-6) M) showed a significant increase in SCEs, but no change in the frequency of G418-resistant revertants. These results suggest that the 2 pathways leading to SCE and recombination respectively are uncoupled, and only a small fraction of the recombination events, if any, are due to unequal SCE in this system.

Frequency and cell specificity of T‐cell receptor interlocus recombination in human cells

Immunoglobulin and T‐cell receptor (TCR) genes are assembled by a site‐specific rearrangement known as V(D)J [variable‐(diversity)‐joining] recombination. These rearrangements occur normally in pre‐B‐ and pre‐T‐cells using signal sequences adjacent to coding exons for immunoglobulin and TCR genes, respectively. However, aberrant recombination may result in the generation of hybrid TCR genes by joining of TCR‐β with TCR‐γ specific sequences. Such hybrid TCR genes occur at a low frequency in peripheral blood lymphocytes (PBL) of healthy individuals, and can be detected by PCR amplification. We have determined the in vivo frequency of hybrid Vγ‐Jβ1 TCR (hybrid TCR) genes in lymphocyte DNA from 12 healthy individuals. The average frequency was found to be 5.83 in 0.75 × 106 PBL, with a threefold difference between the highest and lowest individual value. The presence of similar TCR gene rearrangements in individual samples suggests that T‐cells with a hybrid TCR gene are capable of clonal expansion in vivo. The individual hybrid TCR gene frequency remained relatively constant during 72 hours of in vitro cultivation. In long‐term culture, the frequency gradually decreased, and after 28 days no hybrid TCR genes were detectable in lymphocyte DNA. These results show that T‐cells with a hybrid TCR gene are able to respond to mitogen stimulation in vitro, and may have a proliferative disadvantage or are selected against during prolonged in vitro cultivation. No hybrid TCR genes were detected in ten proliferating T‐cell clones, indicating that the rate of hybrid TCR gene formation is <2.0 × 10−8 per cell per cell division. No hybrid TCR genes were detected in DNA from B‐lymphocytes, sperm, granulocytes, fibroblasts, keratinocytes, and three B‐lymphoblastoid ataxia telangiectasia cell lines. In agreement with previous reports, the frequency of hybrid TCR genes in peripheral blood DNA from two ataxia telangiectasia patients was found to be more than 15‐fold higher than in lymphocytes from normal individuals. These data show that formation of hybrid TCR genes is restricted to T‐cells in vivo, and occurs at a very low frequency, if at all, in proliferating T‐cells in vitro, and with an increased frequency in patients with ataxia telangiectasia. Environ. Mol. Mutagen. 30:245–253, 1997