Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis.

BACKGROUND: The objective of this study was to determine the molecular mechanisms responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. METHODS: Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double-strand break (DSB) repair in cells. Quantitative PCR (Q-PCR) established the expression levels of key DNA DSB repair genes. Imaging flow cytometry using annexin V-FITC was used to compare artemis expression and apoptosis in cells. RESULTS: Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. The Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene, which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Overexpression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. CONCLUSION: We conclude that elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.

ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues.

Clinical and cellular ionizing radiation sensitivity in a patient with xeroderma pigmentosum.

XP14BR is a cell line derived from a xeroderma pigmentosum (XP) patient from complementation group C. The patient was unusual in presenting with an angiosarcoma of the scalp, treated by surgical excision and radiotherapy. Following 38 Gy in 19 fractions with 6 MEV electrons, a severe desquamation and necrosis of the underlying bone ensued, and death followed 4 years later. The cell line was correspondingly hypersensitive to the lethal effects of gamma irradiation. We had previously shown that this sensitivity could be discriminated from that seen in ataxia-telangiectasia (A-T). The cellular response to ultraviolet radiation below 280 nm (UVC) was characteristic of XP cells, indicating the second instance, in our experience, of dual cellular UVC and ionizing radiation hypersensitivity in XP. We then set out to evaluate any defects in repair of ionizing radiation damage and to verify any direct contribution of the XPC gene. The cells were defective in repair of a fraction of double strand breaks, with a pattern reminiscent of A-T. The cell line was immortalized with the vector pSV3neo and the XPC cDNA transfected in to correct the defect. The progeny derived from this transfection showed the presence of the XPC gene product, as measured by immunoblotting. A considerable restoration of normal UVC, but not ionizing radiation, sensitivity was observed amongst the clones. This differential correction of cellular sensitivity is strong evidence for the presence of a defective radiosensitivity gene, distinct from XPC, which is responsible for the clinical hypersensitivity to ionizing radiation. It is important to resolve how widespread ionizing radiation sensitivity is amongst XP patients.

Differential sensitivities of human testicular and bladder tumor cell lines to chemotherapeutic drugs.

The in vitro drug sensitivities of 5 human testicular tumor cell lines (Tera II, SuSa, NEC-8, 833K, T3B1) and 5 human bladder carcinoma cell lines (RT4, RT112, T24, HT1197, HT1376) were compared. Cytotoxicities of cisplatin and doxorubicin were assessed by inhibition of colony-forming ability during continuous exposure to a range of drug concentrations. The ranges of the drug concentrations required to kill 70% of clonogenic cells obtained against the testicular cell lines were 1-7 ng/ml and 21-161 ng/ml for doxorubicin and cisplatin, respectively, compared with 4-19 ng/ml and 112-431 ng/ml for the bladder cell lines. This study shows that continuous cell lines retain the relative clinical chemosensitivities of their tumors of origin. The results also indicate that testicular tumor cells are inherently more sensitive to the cytotoxic effects of chemotherapeutic drugs than are bladder cancer cells.

Inherent sensitivity and induced resistance to chemotherapeutic drugs and irradiation in human cancer cell lines: relationship to mutation frequencies.

Metastatic nonseminomatous testicular germ cell tumors are curable using combination chemotherapy in approximately 80% of patients. In contrast, most other patients with other types of cancer either present with or acquire drug-resistant disease following chemotherapy. Cell lines derived from testis tumors retain hypersensitivity to both drugs and radiation in vitro, thus providing a model system with which to investigate the genetic basis of hypersensitivity to these agents. This study compared the spontaneous and both ethyl methanesulfonate- and cisplatin-induced frequencies of mutation of 6-thioguanine resistance in 3 human bladder and 3 testis tumor cell lines and a bladder and a testis cell line with cisplatin resistance induced in vitro. The two tumor types showed similar frequencies of both spontaneous and induced mutation frequencies at this locus. Therefore, we failed to provide evidence for the hypothesis that the curability of testis tumors is associated with a low frequency of mutation to drug resistance.

The human DNA repair gene, ERCC2 (XPD), corrects ultraviolet hypersensitivity and ultraviolet hypermutability of a shuttle vector replicated in xeroderma pigmentosum group D cells.

To determine the contribution of a human DNA repair gene, ERCC2 (XPD), to mutagenesis in human cells, two ERCC2 (XPD)-transformed xeroderma pigmentosum complementation group D (XPD) cell lines with increased UV survival compared to XP6BE(SV40), the original XPD line, were studied: D6BE-ER2-2 with slightly increased UV survival; and D6BE-ER2-9 with normal UV survival. ERCC2 (XPD) antibody-reactive protein levels were elevated 4.8-fold in D6BE-ER2-2 and 17.6-fold in D6BE-ER2-9 relative to XP6BE(SV40). DNA repair ability was assessed by measuring the ability of the cells to restore expression to UV-treated plasmids. Transfection of pRSVcat exposed to 1000 J/m2 UV resulted in 0.3% chloramphenicol acetyltransferase activity in XP6BE(SV40) cells but 20-80% in D6BE-ER2-2, D6BE-ER2-9, and repair-proficient cells compared to untreated control plasmids. The UV hypersensitivity of the mutagenesis shuttle vector pSP189 in XP6BE(SV40) cells was partially corrected and the UV hypermutability and excess of G:C-->A:T mutations of pSP189 fell to the normal range in D6BE-ER2-2 and D6BE-ER2-9 cells. However, the frequency of plasmids recovered with multiple base substitution mutations was significantly reduced with XP6BE(SV40) cells and remained low in D6BE-ER2-2 and D6BE-ER2-9 cells, when compared with the normal fibroblasts. The human DNA excision repair gene, ERCC2 (XPD), substantially corrected the plasmid UV hypersensitivity and UV hypermutability of xeroderma pigmentosum complementation group D cells; however, the dose response relationship varied for different end points.

Functional evidence of novel tumor suppressor genes for cutaneous malignant melanoma.

Losses of heterozygosity involving chromosomes 9 and 10 are frequent events in the development and progression of cutaneous malignant melanoma. To investigate whether specifically deleted chromosomal regions encode tumor suppressor genes (TSGs), we introduced normal chromosome 10 into the tumorigenic human metastatic melanoma cell line UACC-903 by microcell fusion. In addition, two chromosome 9 derivatives that were microdeleted in the region of the p16INK4A/p15INK4B locus were transferred to determine whether an additional melanoma TSG or TSGs reside on chromosome 9p, as indicated by previous melanoma allele loss studies. In comparison to parental cells, microcell hybrids generated with chromosomes 9 (microdeleted) and 10 displayed reduced anchorage-independent growth in soft agar and markedly reduced tumorigenicity in athymic (nu/nu) mice. These data define a TSG or TSGs that function independently of p15/p16 on chromosome 9 and provide evidence for a TSG (or TSGs) on chromosome 10 that may be important in melanoma development.

Proximal and distal effects of sequence context on ultraviolet mutational hotspots in a shuttle vector replicated in xeroderma cells.

Hotspots are a standard feature of mutational spectra induced by mutagens in a variety of marker genes. While it is generally believed that sequence context exerts an important influence on hotspot location, direct experimental evidence is quite limited. We have studied ultraviolet mutagenesis in a suppressor tRNA marker gene (supF) carried in a mammalian shuttle vector and replicated in Xeroderma pigmentosum cells in culture. We have now constructed a small family of functional variant suppressor tRNA marker gene which differ from one another by one or two nucleotide changes. UV mutational spectra were generated for each variant gene. We found that the change of a dipyrimidine from 5' TC to 5' CC eliminated a strong mutational hotspot. In addition a single base change in the supF gene was accompanied by the appearance of a new hotspot eight bases away. Finally, another single base change suppressed a major hotspot 48 bases away. Polymerase stop assays on the UV modified marker genes were used to measure the frequency and distribution of photoproducts. The differences in hotspot patterns could not be explained by differences in modification patterns. These results indicate that a change in sequence context can directly influence the probability of mutagenesis at specific sites.

Xeroderma pigmentosum and related disorders: examining the linkage between defective DNA repair and cancer.

Xeroderma pigmentosum, Cockayne syndrome, the xeroderma pigmentosum-Cockayne syndrome complex, and trichothiodystrophy cells have defects in DNA repair and are associated with clinical and cellular hypersensitivity to ultraviolet radiation (UV). Familial dysplastic nevus syndrome cells have UV hypermutability. Although xeroderma pigmentosum and dysplastic nevus syndrome have markedly increased cancer risk. Cockayne syndrome and trichothiodystrophy do not. At the molecular level, these disorders are associated with several different genetic defects as evidenced by the existence of multiple overlapping complementation groups. Recent progress has been made in identifying the chromosomal location and cloning the defective genes in these disorders. Using plasmid shuttle vectors we have shown abnormal repair and mutagenesis of DNA damaged by 254-nm (UVC) or 295-nm (UVB) radiation or the chemical carcinogen aflatoxin in cells from patients with xeroderma pigmentosum. Although xeroderma pigmentosum cells are defective in repair of all photoproducts, Cockayne syndrome cells appear to be defective in repair of cyclobutane dimers and have normal repair of nondimer photoproducts. DNS cells have post UV plasmid hypermutability. These diseases may serve as models for examining molecular mechanisms of carcinogenesis in humans.

A signature element distinguishes sibling and independent mutations in a shuttle vector plasmid.

We have developed a new shuttle vector plasmid for studying mutagenesis in mammalian cells that permits proof of independence of identical mutations. Mutations occur more frequently at some sites in a gene than in others, and in a collection of mutant plasmids from a single transfection of mammalian cells the same mutation may appear several times. However, those arising from independent events cannot be distinguished from siblings of an initial event. The new vector system (pSP189) is a population of plasmids, each of which contains an 8-bp 'signature sequence'. This sequence confers a unique identification tag to each plasmid and allows individual members to be identified by a distinctive signature. The plasmid also carries the Escherichia coli bacterial supF gene as a marker for mutagenesis, as well as sequences which support replication in primate (including human) cells and E. coli. We have used the pSP189 system to generate a UV-induced spectrum of mutations in supF following replication in a single plate of human DNA-repair-deficient cells (xeroderma pigmentosum, complementation group A). With the signature sequence, we were able to determine whether identical mutations derived from the transfection were of independent or sibling origin. There were eight identical mutations at the strongest hotspot, all of which had different signature sequences. Only one of these events would have been reported in previous experiments. This plasmid reduces the effort required to generate a spectrum of mutations caused by a DNA-damaging agent and allows a more accurate assessment of mutational hotspot intensity.

pSV3neo transfection and radiosensitivity of human cancer cell lines.

Immortalisation of human fibroblasts by transfection with a plasmid, pSV3neo, results in an increase in their radioresistance. The change in radiosensitivity may either be a consequence of transformation or due to expression of the SV40 T-antigen in pSV3neo. To investigate these two possibilities, we transfected pSV3neo into cells already transformed and immortalised. The radiosensitivities of three human bladder cancer cell lines were unaltered in clones expressing T-antigen, indicating that the changes observed in fibroblasts probably are a consequence of transformation, and not the presence of SV40 T-antigen.

Telomerase activity in the human breast.

Telomerase is a ribonucleoprotein enzyme which appears to play an important role in carcinogenesis. Telomerase reactivation seems to be associated with immortalization and malignancy. Using a PCR-based assay, we examined telomerase activity in 50 breast tissue specimens, prospectively obtained from 37 women undergoing elective breast surgery. The specimens examined included normal breast (n=13), benign breast lesions (n=5), ductal carcinoma in situ (n=8) and infiltrating ductal carcinoma (n=24). All normal breast, benign breast and DCIS specimens lacked telomerase activity. Sixteen (67%) of 24 infiltrating carcinomas. In infiltrating ductal cancer, there was a statistically significant association between telomerase activity and nodal metastasis. The present results indicate that telomerase activity is associated with acquisition of invasive malignancy in the human breast and may have a role in complementing cytopathological diagnosis. Telomerase activity as a prognostic marker should be included in future validation studies. In DCIS, telomerase activity may be a late event associated with invasion of the basement membrane.

Telomerase activity in phyllodes tumours.

AIMS: To investigate telomerase, a ribonucleoprotein that synthesizes telomeres. Recent evidence suggests that telomerase reactivation is associated with the acquisition of immortalization and malignancy. METHODS: Using a sensitive PCR-based assay (the TRAP assay), we examined telomerase activity in two recurrent phyllodes tumours in two patients. RESULTS: Both tumours expressed telomerase activity. Histological examination of the lesions, according to the criteria proposed by Azzopardi and Salvadori, revealed a malignant phyllodes tumour in one patient and a benign phyllodes tumour in the other patient. CONCLUSIONS: Our findings suggest that telomerase activity may have a potential role as a prognostic marker in predicting the clinical behaviour of these rare tumours.

Telomerase activity and lymphovascular invasion in breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that plays an important role in cell immortalization and carcinogenesis. Lymphovascular invasion (LVI) is a fundamental step in the process of breast cancer metastasis and is recognized as an important prognostic factor in patients with breast cancer. METHODS: Using a PCR-based assay, telomerase activity was determined in 34 prospectively collected infiltrating breast carcinomas. Adjacent sections of the specimens were examined histologically by two experienced breast pathologists using light microscopy and haematoxylin & eosin staining. RESULTS: Telomerase activity was detected in 24 (71%) of 34 breast tumours. Two (20%) of 10 telomerase-negative tumours had LVI compared with 14 (58.3%) of 24 telomerase-positive tumours. This association was statistically significant (P<0.05). Telomerase activity was also significantly associated with nodal metastases but not with tumour grade, tumour size or menopausal status. CONCLUSIONS: Telomerase reactivation is significantly associated with LVI in breast cancer and may reflect the metastatic potential of the disease.

Telomerase activity and prognosis in breast cancer.

AIMS: Telomerase is a ribonucleoprotein enzyme which appears to play an important role in carcinogenesis. Its reactivation is associated with the acquisition of immortalization and malignancy. The present study aims to examine the association between telomerase activity and prognosis in breast cancer. METHODS: Using a PCR-based assay, we retrospectively examined telomerase activity in 45 frozen human breast cancer specimens. Telomerase activity was compared with histopathological and clinical data. RESULTS: Telomerase activity was detected in 20 (44%) of 45 cases and was associated with advanced histopathological grade and tumour type (ductal vs. lobular). The association with these histological parameters was statistically significant (chi-squared test P<0.05). There was no significant difference in the overall survival rate (78 vs. 77%) or disease-free survival (73 vs. 69%) at 5 years (Kaplan-Meier method, log-rank test P>0.05). CONCLUSIONS: The present results indicate that telomerase activity in human breast cancer is not associated with nodal status or disease outcome.

The association between telomerase, histopathological parameters, and KI-67 expression in breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that appears to play an important role in carcinogenesis. Telomerase reactivation seems to be associated with immortalization and malignancy. METHODS: Using a polymerase chain reaction (PCR)-based assay known as the TRAP (telomeric repeat and amplification protocol) assay, we examined telomerase activity in 60 breast specimens prospectively collected from 39 patients undergoing elective breast surgery in our center. The specimens included adjacent noncancerous breast (n = 21), benign breast disease (n = 5), and infiltrating carcinoma (n = 34). Ki-67 expression was determined in 32 invasive breast cancer specimens using immunohistochemistry techniques. The histopathological features were determined by light microscopy by an experienced breast pathologist. RESULTS: Telomerase activity was detected in 24 (71%) of 34 infiltrating carcinomas. None of the adjacent noncancerous specimens nor the benign breast lesions expressed telomerase activity. Telomerase reactivation was significantly associated with nodal metastasis and Ki-67 expression. There was no significant association between telomerase activity and menopausal status, tumor grade, or tumor size. CONCLUSIONS: Telomerase reactivation is associated with the acquisition of malignancy in the human breast. Telomerase activity is significantly associated with nodal metastasis and cellular proliferation as measured by Ki-67 expression in human breast cancer.

Complete non-puerperial uterine inversion as a result of a uterine sarcoma.

Complete non-puerperial uterine inversion is rare and when present is usually associated with a prolapsed submucous fibroid. The inversion in this case was associated with a uterine sarcoma in an 88 year old diabetic patient, gravida 13, who presented with a four month history of intermittent vaginal bleeding. She was successfully managed with a total abdominal hysterectomy and some of the difficulties with diagnosis and management are highlighted.

Ultraviolet mutagenesis in human lymphocytes: the effect of cellular transformation.

We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells. To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189. Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058). pZ189 was treated with uv and introduced into the four cell lines by electroporation. Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli. Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines. Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%). In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells. Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared. Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia.

Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal.

We compared the contribution to mutagenesis in Cockayne syndrome (CS) cells of the major class of UV photoproducts, the cyclobutane pyrimidine dimer, to that of other DNA photoproducts by using the mutagenesis shuttle vector pZ189. Lymphoblastoid cell lines from the DNA repair-deficient disorders CS and xeroderma pigmentosum (XP) and a normal line were transfected with UV-treated pZ189. Cyclobutane dimers were selectively removed before transfection by photoreactivation (PR), leaving nondimer photoproducts intact. After UV exposure and replication in CS and XP cells, plasmid survival was abnormally reduced and mutation frequency was abnormally elevated. After PR, plasmid survival increased and mutation frequency in CS cells decreased to normal levels but remained abnormal in XP cells. Sequence analysis of > 200 mutant plasmids showed that with CS cells a major mutational hot spot was caused by unrepaired cyclobutane dimers. These data indicate that with both CS and XP cyclobutane dimers are major photoproducts generating reduced plasmid survival and increased mutation frequency. However, unlike XP, CS cells are proficient in repair of nondimer photoproducts. Since XP but not CS patients have a high frequency of UV-induced skin cancers, our data suggest that prevention of UV-induce skin cancers is associated with proficient repair of nondimer photoproducts.

Intravesical chemotherapy: combination with dimethyl sulfoxide does not enhance cytotoxicity in vitro.

There is evidence that dimethyl sulfoxide (DMSO) can increase the anticancer activity of chemotherapeutic drugs. As DMSO is instilled into the bladder for interstitial cystitis, it could be readily adopted in clinical practice if it was found to enhance the effectiveness of the drugs used for intravesical chemotherapy. The purpose of this study was to investigate, using a human bladder cancer cell line, the hypothesis that DMSO enhances the activity of these agents. However, the addition of 4% DMSO to the four drugs most frequently used for intravesical chemotherapy (adriamycin, epodyl, mitomycin-c, thiotepa) did not increase tumour cell kill in vitro.