Tumorigenic conversion of xeroderma pigmentosum lymphoblastoid cells without karyotypic alteration

In order to examine the process of malignant transformation of human somatic cells, we studied the tumorigenic conversion of an Epstein-Barr-virus-immortalized lymphoblastoid cell line (LCL) derived from a patient with xeroderma pigmentosum (XP) complementation group A. Repeated irradiation of the XP cells, XP7NI, with UV-light and subsequent treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the acquisition of tumorigenicity in athymic nude mice. The tumorigenicity of XP7NI cells was also induced by TPA treatment alone. The tumors formed in athymic mice were of B-cell lymphoma with characteristic histology, cell surface immunoglobulins and an antigen as detected by a B-cell-specific monoclonal antibody (MAb), CD20. The surface immunoglobulins and the HLA type of these tumor cells were identical with those of the parental cells. These malignantly transformed cells retained the same UV sensitivity, serum requirement, colony-forming ability in soft agar, and normal human karyotype as the parental cells. Unlike other tumorigenic lymphoblastoid cell lines, this XP lymphoblastoid cell line provides a unique case in that process(es) leading to tumorigenicity may be induced by UV and TPA without apparent karyotypic changes.     

Mutagenesis and lethality following S phase irradiation of xeroderma pigmentosum and normal human diploid fibroblasts with ultraviolet light

The mutagenic and lethal effects of u.v. light exposure in theDNA synthetic phase of the cell cycle were determined in xerodermapigmentosum complementation group A (XP-A), hereditary adenomatosisof the colon and rectum (ACR), and a normal, foreskin derivedcell strain (AG1522). For AG1522, an increased sensitivity tothe cytotoxic effects of u.v. light (survival curve D₀ = 3.2J/m²) was observed as compared to previous findings for confluent,non-proliferating cultures (D₀ = 4.2 J/m²). XP-A fibroblastswere markedly hypersensitive (D₀ = 0.5 J/m²) and ACR fibroblastsexhibited an intermediate response (D₀ = 2.0 J/m²). The mutagenicresponse of ACR fibroblasts, however, was similar to normalfibroblasts. A threshold of 1.5–2 J/m² was observed foru.v. induced mutagenesis in normal and ACR fibroblasts. XP fibroblasts,on the other hand, were strikingly hypermutable and demonstratedlittle or no threshold. When S phase mutagenesis was consideredas a function of survival level rather than u.v. light dose,XP fibroblasts remained significantly hypermutable as comparedwith normal fibroblasts at all survival levels. Previous mutagenesisresults with confluent, nonproliferating cultures of XP andnormal fibroblasts were reanalyzed as a function of cytotoxicity;XP hypermutability at all survival levels was also observed.     

A truncated human xeroderma pigmentosum complementation group A protein expressed from an adenovirus sensitizes human tumor cells to ultraviolet light and cisplatin

Individuals with the genetic disease xeroderma pigmentosum (XP) have impaired nucleotide excision repair (NER). Group A XP cells are defective in the XPA protein essential for NER and serve, together with other NER proteins, as a nucleation factor for the demarcation of bulky DNA damage. Because XPA cells are extremely sensitive to UV and drugs that cause bulky DNA damage, the XPA protein is an attractive target for manipulating cellular sensitivity to certain cancer therapeutics, a concept that perhaps can be applied toward developing more effective cancer treatments. We have made a replication-defective adenovirus, AdCMV-FlagXPA(59-114), that expresses a truncated form of XPA encompassing amino acids 59-114 sufficient for binding to the excision repair cross-complementing protein 1 (ERCC1)/xeroderma pigmentosum complementation group F (XPF) nuclease essential for making an incision 5' of the damage. On the basis of previous work, it was expected that this truncated XPA protein would work as a decoy and impair NER and, thus, sensitize cells to UV and drugs that produce bulky DNA lesions. Because the truncated XPA protein is "tagged" with the Flag epitope, an anti-Flag antibody can be used to detect protein expression and to isolate proteins associated with the XPA complex. We show that relatively large quantities of truncated XPA protein are present in infected human lung carcinoma A549 cells 2-4 days postinfection. Moreover, in a pull-down assay using anti-Flag antibody, we show that ERCC1 is present in the FlagXPA complex but not in a complex isolated from cells infected with a control virus. Most importantly, cells infected with AdCMV-FlagXPA(59-114) are significantly more sensitive than control cells to UV-induced damage as determined by host-cell reactivation of UV-irradiated AdLacZ adenovirus and in a cytotoxicity assay that appears to be the result of aberrant processing of 6-4 photoproducts. Infected cells were also more sensitive to treatment with cisplatin, an important cancer drug. These results suggest that NER, and the XPA protein in particular, can be a direct target for sensitizing tumor cells to UV and cisplatin and perhaps also certain other clinically important drugs.     

Abnormal sensitivity of human fibroblasts from xeroderma pigmentosum variants to transformation to anchorage independence by ultraviolet radiation

One class of xeroderma pigmentosum (XP) patients, known as XP variants, inherit the characteristic predisposition to sunlight-induced skin cancer, but unlike the majority of XP patients, their cells do not exhibit a deficiency in rate of excision repair of ultraviolet (UV) radiation-induced DNA damage. XP variant cells are only slightly more sensitive than normal to killing by 254 nm UV radiation or simulated sunlight. But they are much more sensitive than normal to the induction of mutations by these agents. We investigated their sensitivity to UV-induced transformation to anchorage independence compared to that of normal cells. Low doses of UV (2 to 4.5 J/m2), doses which resulted in little or no measurable transformation in normal cells, caused a dose-dependent increase in the frequency of anchorage independent XP variant cells. Doses of 6 to 8 J/m3 were required to elicit a comparable response in the normal fibroblasts. Even when the two kinds of cells were compared at doses adjusted to give equal cytotoxicity, the frequency of transformation in the XP variant cells was higher than normal. Thus, their sensitivity to induction of anchorage independence by UV paralleled their sensitivity to UV-induced mutations.     

A convenient method of establishing permanent lines of xeroderma pigmentosum cells.

Nine lymphoblastoid cell lines were established after transformation by Epstein-Barr virus of peripheral lymphocytes from four xeroderma pigmentosum (XP) patients, the parents of one XP patient, and three normal donors. All these cell lines proliferate as suspension in Roswell Park Memorial Institute Medium 1640 supplemented with 20% fetal bovine serum, without detectable release of infectious Epstein-Barr virus. Some characteristics of these cell lines, such as growth rates, chromosome numbers, UV sensitivities, and activities of unscheduled DNA syntheses induced by UV, 4-nitroquinoline 1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine, were determined. Results confirm that the properties related to XP are not altered by transformation with Epstein-Barr virus and are the same in degrees of defect as are those of dermal fibroblasts from the respective individuals. These XP and normal lymphoblastoid cell lines should be especially useful for biochemical studies on the mechanism of DNA repair, because they are easy to grow in mass culture.     

Increased ultraviolet sensitivity and chromosomal instability related to P53 function in the xeroderma pigmentosum variant

The xeroderma pigmentosum (XP) variant (XPV) is a form of XP that has normal excision repair but shows defective DNA replication after UV irradiation. In developing various transformed fibroblast cell lines from these patients, we have found that there are significant phenotypic changes in transformed cells that seem to correlate with inactivation of p53. After transformation with SV40, XPV cell lines are only slightly UV sensitive, like their primary counterparts, but their sensitization with caffeine and the induction of sister chromatid exchanges (SCEs) by UV irradiation are greatly enhanced. After transformation by HPV16 E7, which targets the retinoblastoma cell cycle regulatory gene, there is no change in the UV sensitivity of XPV cells; but, when transformed by HPV16 E6 or E6 and E7 combined, there is a large increase in UV sensitivity and in the induction of SCEs. These changes are not associated with any detectable changes in the reactivation of an externally irradiated luciferase expression vector, the excision of cyclobutane pyrimidine dimers from bulk DNA, or unscheduled DNA synthesis and, therefore, do not involve excision repair. We suggest that if SCEs represent homologous recombination between sister chromatids, then in the absence of p53 function, the DNA chain arrest typical of UV-damaged XPV cells initiates strand exchange during recovery. In untransformed cells with normal p53, the preferred mode of recovery would then be replication bypass. The symptoms of elevated solar carcinogenesis in XPV patients may, therefore, be associated with increased genomic instability in cells of the skin in which p53 is inactivated by UV-induced mutations.     

Abnormal response of xeroderma pigmentosum cells to bleomycin

The repair of bleomycin-damaged DNA was examined in human fibroblasts isolated from patients having the disease xeroderma pigmentosum (XP). In normal fibroblasts, the appearance of low-molecular-weight DNA was observed in the presence of increasing amounts of the drug. The studies in XP fibroblasts produced results which differed from those obtained in normal cells in two ways. (a) Prelabeled XP cells from most complementation groups contained more low-molecular-weight DNA than observed in the other human fibroblasts examined. (b) When XP cells were exposed to low doses of bleomycin, the low-molecular-weight DNA disappeared, suggesting induction of a repair process. If the XP cells were exposed to bleomycin in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, the disappearance of low-molecular-weight DNA was not observed; instead, a normal dose response to the drug was observed. Our results suggest that XP cells show an "induced" repair response following bleomycin treatment and that blocking DNA chain elongation uncovers normal incisions in bleomycin-treated DNA.     

Enhanced transformation of xeroderma pigmentosum variant cells by ultraviolet light-irradiated simian virus 40.

The role of DNA repair in transformation was investigated by infecting repair-deficient xeroderma pigmentosum (XP) variant cells, XP variant heterozygous cells, and normal human fibroblasts with simian virus 40 which had been irradiated by ultraviolet light. The transformation frequencies obtained were compared to those observed for unirradiated virus. While normal and heterozygous cells showed no differences between transformation frequencies using either irradiated or untreated virus, two XP variant cell lines were transformed 2- to 7-fold more readily with irradiated virus than with unirradiated virus. XP variant cells were also found to produce lower than normal quantities of virus following infection with either damages or undamaged virus, suggesting that increased viral production was not contributing to the increased transformation seen for these cells. Finally, the proportion of cells which repair ultraviolet light-irradiated simian virus 40 was found to be similar for wild-type and XP variant cells, suggesting that enhanced transformation in the mutant cells was not associated with a reduction in the numbers of cells which repair damaged virus. Several possible mechanisms to account for the increased transformation of XP variant cells by ultraviolet light-irradiated simian virus 40 are proposed.     

Re-exposure of human lymphoblastoid cell lines to Epstein-Barr virus

Human lymphoblastoid lines of various origins which harbour Epstein-Barr virus (EBV)-specific nucleic acid were re-exposed to EBV. Following infection, cells of the non-virus-producing lines, Raji and S 95, predominantly synthesized EBV-specific early antigens (EA), whereas only a small percentage of cells revealed viral capsid antigens (VCA). In Raji cells, the number of VCA-producing cells was paralleled by the percentage of virus-specific DNA-synthesizing cells. In S 95 cells, however, viral DNA-synthesizing cells exceeded the number of VCA-producing cells by a factor of more than 10. Induction of EA in Raji cells was dose-dependent and inversely related to cell growth. Irradiation of the virus by ultraviolet light prior to infection led to reduced infectivity. This reduction seemed to follow single-hit kinetics. Raji cells, previously re-exposed to EBV, showed reduced EA induction after re-infection with EBV, as compared to Raji control cells not previously exposed. Of 10 lines which spontaneously synthesize EBV-specific antigens, seven lines proved to be refractory to re-infection, whereas three were as susceptible as the Raji and S 95 controls. From three of the refractory lines infectious virus could be recovered from the culture medium prior to infection. These results permit the following interpretations: (1) the response of human lymphoblastoid cells after re-infection with EBV results from the infecting virus and not from stimulation of endogenous genomes; (2) cells demonstrating EA synthesis ultimately die; (3) re-exposure to EBV increases the resistance to re-infection of the surviving cells; and (4) cell lines producing infectious EBV are refractory to re-infection. It is suggested that the spontaneous synthesis of infectious virus favours the selection of resistant cells.     

Progress of research on xeroderma pigmentosum

Xeroderma pigmentosum (XP) is an autosomal recessive human disease, clinically characterized by high incidence of skin cancers on sun-exposed areas. XP cells are hypersensitive to killing by ultraviolet light (UV), because they have a defect in DNA excision repair of UV-induced DNA damages. Genetic complementation analysis by cell fusion has identified 9 genetic complementation groups, designated groups A through H and a variant. However, the genetic basis of the physiological defect of XP has not yet been characterized. Recently, XP genes and human DNA repair genes have been molecularly cloned by DNA transfection methods. Molecular biological analysis of these genes should be a clue to elucidating the molecular mechanism of DNA repair in human. Moreover, an in vivo microinjection system and an in vitro system for study of DNA repair synthesis promoted by human cell extract have been developed and they can be utilized as assays during the purification of protein factors that complement repair defective XP cells. A nuclear factor that binds to DNA lesion has been identified and it was defective in group E XP cells. Yeast homolog of this nuclear factor appears to be a photolyase.     

Enhanced cell killing and mutagenesis by ethylnitrosourea in xeroderma pigmentosum cells

It was shown previously that following treatment with ethylnitrosourea(ENU), SV40-transformed cells from xeroderma pigmentosum patientXP12RO, complementation group A remove O⁶-ethylguanine fromtheir DNA 2 to 3 times more slowly than do such cells from anormal individual (GM637), but that each of the several otherDNA adducts studied are removed at the same rate by both celllines. If O⁶-ethylguanine contributes significantly to the cytotoxiceffect of ENU, a population which can excise this lesion morerapidly than another during the posttreatment period shouldexhibit a higher survival. We compared these two cell linesfor survival of colony-forming ability following exposure toENU. XP12RO-SV40 cells proved 3.5-fold more sensitive thanGM637. To extend this study to non-transformed diploid humanfibroblasts, we compared the survival of cells derived fromnormal individuals (NF) with that of fibroblasts from severalXP patients: XP12BE (group A), XP7BE (group D), and XP4BE (anXP variant). The survival curve of NF, XP7BE, and XP4BE cells,but not XP12BE cells, exhibited a distinct shoulder. The slopeof the survival curve for XP12BE cells was 3-fold steeper thanthat of the exponential part of the curve for NF; the slopeof the curve for XP7BE cells, 2-fold steeper; and that of XP4BEcells, 1.7-fold steeper. This enhanced cytotoxicity was notobserved in XP12BE cells treated with methylating agents suchas methylnitrosourea or N-methyl-N' -nitro-N-nitroso-guanidine.We compared NF and XP12BE cells for sensitivity to the mutagenicaction of ENU and found 3-fold higher frequencies of 6-thioguanineresistant mutants in the XP cells. This result is expected ifthe XP cells are slower than normal in excising O⁶-ethylguanineand if this adduct is mutagenic. When we compared the frequencyof mutations induced by ENU and u.v. radiation at doses whichcaused equal cell killing, ENU was 4-fold more efficient a mutagenthan u.v.     

Stimulated production of ATP by H2O2 disproportionation in extracts from normal and xeroderma pigmentosum skins, and from normal, xeroderma pigmentosum, ataxia telangiectasia and simian virus 40 transformed cell lines

It has been previously shown that xeroderma pigmentosum (XP) skin biopsies and their established cell lines exhibit a decrease in catalase activity and enhanced formation of photo-produced H2O2. Several in vivo and in vitro thermodynamic results suggest that the energy of H2O2 disproportionation produced by catalase could be sufficient to synthesize ATP with or without the help of intact mitochondria. In this paper, we first studied the properties of H2O2-stimulated ATP production in extracts of normal and pathological XP skin biopsies and cell lines. In acellular extracts of normal skin biopsies and/or cell lines, ATP production can be increased 2- to 3-fold, but only with a narrow range of H2O2 concentration. In contrast, in extracts of pathological skins or cells, ATP production was only observed when using 10- to 1000-fold less H2O2 concentration as defined for normal extracts. Similar results were noted with two cell lines derived from patients afflicted with ataxia telangiectasia (AT), and with simian virus 40 (SV40) transformed lines of normal, XP and AT cells, Although we have no proof that such a process may exist in vivo, we would like to suggest that both H2O2-stimulated ATP production and catalase activity are good indicators of the degree of normality or abnormality of skin biopsies and/or cell lines.     

Decreased calcium dependence of lymphoblastoid cell lines compared with Burkitt lymphoma cell lines

The extracellular calcium level required for proliferation was compared in B lymphoid cell lines from various sources by determining the calcium concentration at which long-term proliferation was inhibited by 50% (CaPD50). Fourteen Burkitt lymphoma (BL) lines had a mean CaPD50 of 44 +/- 28 microM whereas 45 lymphoblastoid cell lines (LCLs) obtained by in vitro transformation of B lymphocytes with Epstein-Barr virus (EBV) had a mean CaPD50 of 3.6 +/- 1.8 microM. This difference applied also to autologous BL lines and LCLs established from the same patient. The decreased calcium requirement of virally-transformed compared with tumour-derived cell lines therefore appears to be a universal phenomenon in mammalian cells. Within the BL group, no correlation was found between the calcium requirement for proliferation and presence or absence of the EBV genome. Arrest of BL lines and LCLs occurred in the G1 phase of the cell cycle and was readily reversed by addition of calcium to the medium. One anomalous LCL was found which showed a high CaPD50 (43 +/- 6 microM) and accumulated in both G1 and G2. These results, in combination with a previous study of EBV transformation in vitro, indicate that the calcium dependence of B lymphocytes generally decreases in the following order: normal cells greater than BL cells = early stage transformation greater than LCL. The 2 transformed phenotypes thus distinguished in human lymphoid cells may offer unique opportunities for defining the status and expression of EBV in vitro and in vivo.     

Retinoid-induced growth inhibition of herpesvirus-transformed marmoset lymphoblastoid cell lines

The in vitro proliferation of herpesvirus-transformed marmoset lymphoblastoid cell lines (LCL) was inhibited by retinoic acid and retinyl acetate. Both Herpesvirus-ateles-and Herpesvirus-saimiri-transformed LCL with T-cell characteristics and Epstein-Barr-transformed LCL with B-cell markers were more sensitive to retinoic acid than to retinyl acetate. Inhibition of LCL proliferation was dependent on retinoid concentration and became apparent only after 3-4 days of exposure. In vitro assays for marmoset LCL sensitivity to retinoids may indicate the potential usefulness of these compounds in the chemoprevention of virus-induced lymphoma in vivo.     

Repair of DNA lesions induced by ultraviolet irradiation and aromatic amines in normal and repair-deficient human lymphoblastoid cell lines

A host cell reactivation (HCR) assay was employed to study thecapacity of a normal and three repair-deficient human lymphoblastoidcell lines to repair DNA damage induced by UV irradiation andthe aromatic amines 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine(PhIP) and N-acetyl-2-aminofluorene (AAF) respectively. Thecell line belonging to xeroderma pigmentosum complementationgroup C (XP-C) removed all three types of damage less efficientlythan the normal cell line, but more efficiently than the cellline belonging to xeroderma pigmentosum complementation groupD (XP-D). The cell line belonging to complementation group Bof Cockayne's syndrome (CS-B) showed reduced host cell reactivation.Fibroblasts from CS-B patients have reduced gene-specific DNArepair, but normal total genomic DNA repair, thus our data suggestthat the HCR assay measures the capacity for gene-specific DNArepair. In the XP-D cell line, which has practically no DNArepair capacity, AAF adducts had a more potent inhibitory effecton gene expression than UV and PhIP adducts. When correctedfor this inhibitory effect, the wild-type, XP-C and CS-B celllines repaired low levels of AAF and UV adducts with similarefficiencies, however, PhIP adducts were repaired less efficiently.