Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro

Sun exposure has been clearly implicated in premature skin aging and neoplastic development. These features are exacerbated in patients with xeroderma pigmentosum (XP), a hereditary disease, the biochemical hallmark of which is a severe deficiency in the nucleotide excision repair of UV-induced DNA lesions. To develop an organotypic model of DNA repair deficiency, we have cultured several strains of primary XP keratinocytes and XP fibroblasts from skin biopsies of XP patients. XP skin comprising both a full-thickness epidermis and a dermal equivalent was successfully reconstructed in vitro. Satisfactory features of stratification were obtained, but the expression of epidermal differentiation products, such as keratin K10 and loricrin, was delayed and reduced. In addition, the proliferation of XP keratinocytes was more rapid than that of normal keratinocytes. Moreover, increased deposition of cell attachment proteins, alpha-6 and beta-1 integrins, was observed in the basement membrane zone, and beta-1 integrin subunit, the expression of which is normally confined to basal keratinocytes, extended into several suprabasal cell layers. Most strikingly, the in vitro reconstructed XP skin displayed numerous proliferative epidermal invasions within dermal equivalents. Epidermal invasion and higher proliferation rate are reminiscent of early steps of neoplasia. Compared with normal skin, the DNA repair deficiency of in vitro reconstructed XP skin was documented by long-lasting persistence of UVB-induced DNA damage in all epidermal layers, including the basal layer from which carcinoma develops. The availability of in vitro reconstructed XP skin provides opportunities for research in the fields of photoaging, photocarcinogenesis, and tissue therapy.     

Xeroderma pigmentosum patients from the federal Republic of Germany: Decrease in post-UV colony-forming ability in 30 xeroderma pigmentosum fibroblast strains is quantitatively correlated with a decrease in DNA-incising capacity

Summary A total of 16 normal and 46 XP fibroblast strains from the Mannheim Collection were investigated for colony-forming ability following exposure to both UV light and the UV-like carcinogen (Ac)₂ONFln. The dose-response experiments included up to 13 dose levels. The exponential segments of the curves were analysed by linear regression and the negative reciprocal of the regression coefficient (D₀) was calculated for each cell strain.For quantitating the DNA-incising capacity, DNA elution curves were determined at several UV dose levels. Plotting the initial velocities of the elution curves versus the UV dose yielded a regression line, the slope of which was used to obtain the characteristic value E₀.Comparing D₀ with E₀ values showed that cell strains in which colony-forming ability was reduced suffered a reduction of DNA-incising capacity of the same magnitude. There were only 3 exceptional strains in which reduction of DNA-incising capacity was less pronounced than reduction of colony-forming ability. We have previouly shown (Fischer et al. 1982) that D₀ values from 27 XP strains of the Mannheim Collection were correlated with clinical symptoms. This correlation is now being extended by relating colony-forming ability to the magnitude of the DNA incision defect. From our data we conclude that the best quantitative biochemical denominator to explain the sun sensitivity of XP is that of a defective incision of UV-damaged DNA.A considerable similarity in sensitivity towards both UV light and (Ac)₂ONFln was found in 16 normal and 46 XP strains. This seems to indicate that UV-and (Ac)₂ONFln-induced DNA damage are removed to a large extent by the same pathways in human fibroblasts.Abbreviations XP xeroderma pigmentosum - (Ac)₂ONFln N-acetoxy-2-acetylaminofluorene - UV light ultraviolet light - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - ara-C 1--d-arabinofuranosyl cytosine This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

Doxorubicin and γ rays increase the level of DNA topoisomerase IIα in nuclei of normal and xeroderma pigmentosum fibroblasts

DNA topoisomerase IIα was monitored with the monoclonal antibody Ki-S1 in human fibroblasts after irradiation of cells with γ rays from a ¹³⁷Cs source or treatment with the DNA topoisomerase II inhibitor doxorubicin. DNA topoisomerase IIα was localized immunohistochemically as bright fluorescent dots in the karyoplasm. The fibroblasts investigated originated from normal human donors and a xeroderma pigmentosum (XP) patient (XP12BE). All cell lines examined showed a time- and dose-dependent increase in DNA topoisomerase IIα abundance after irradiation or treatment with doxorubicin. No principal difference in response was seen between normal and XP fibroblasts towards either treatment alone. After irradiation with 9 Gy, the effect was detectable after as little as 30 min and lasted for at least 6 h. After doxorubicin treatment, topoisomerase II overexpression occurred within less than 2 h. It passed through a maximum and began to decrease after approximately 6 h. In principle, the increase in DNA topoisomerase IIα may result from (i) architectural changes of interphase chromatin leading to enhanced accessibility of preformed enzyme to the antibody, (ii) enhanced gene expression, or (iii) enhanced stabilization of mRNA or protein molecules. The increase in enzyme levels may be part of the well-known DNA damage responses that operate in cell-protective or DNA-reparative pathways. Thus, the action of DNA topoisomerase II would serve to catalyze preparatory steps in DNA repair. We also found overexpression of the Bax protein and p16 predominantly in treated XP cells, suggesting that the DNA-damaging protocols elicited signals for apoptosis and cell-cycle arrest. From the simultaneous increase in DNA topoisomerase IIα and Bax, one may conclude that DNA topoisomerase IIα also plays role in apoptosis. Received: 10 February 1998 / Accepted: 18 March 1998     

6-Methylguanine and 6-methylguanosine inhibit colony-forming ability in a malignant xeroderma pigmentosum cell line but not in other xeroderma pigmentosum and normal human fibroblast strains after treatment with 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)-urea

Summary The XP cell strain XP29MA, its malignant counterpart XP29MAmal and a normal human fibroblast strain were tested for colony-forming ability after treatment with HECNU in the presence of m⁶G, m⁶Gua, and he⁷G.In XP29MAmal, inhibition of post-HECNU colony-forming ability was 35% when 0.25 mM of either m⁶G or m⁶Gua were present, whereas in XP29MA and the normal fibroblast strain no inhibition was detected. The he⁷G caused a similar but smaller inhibitory effect in XP29MAmal, but failed to do so in XP29MA.HECNU predominantly exerts its killing effect by alkylating O-6 of DNA-bound guanine and causing DNA interstrand crosslinks. Alkylation of O-6 of guanine can be repaired by 6-methylguanine-DNA methyltransferase. From our experiments we conclude that in XP29MAmal this methyltransferase was inhibited in the presence of the 6-alkylguanines, thus leaving more 2-chloroethylated sites in DNA unrepaired. This results in sensitization in terms of decreased colony-forming ability observed only in the malignant cell line.Abbreviations XP xeroderma pigmentosum - HECNU 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)-urea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - m⁶G 6-methylguanosine - m⁶Gua 6-methylguanine - he⁷G 7-(2-hydroxyethyl)-guanosine This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136The publication is dedicated to Professor E. Hecker on the occasion of his 60th birthday     

Gene transduction into murine primitive hematopoietic cells with 2-gene retroviral vectors using a Transwell coculture system

The present study aims at expressing a reporter gene in hematopoietic cells in vivo by introducing it into primitive hematopoietic cells with a 2-gene retroviral vector. Various constructs of retroviral vectors containing the human IL-2 receptor α chain gene (TAC) as the reporter and the neomycin phosphotransferase gene (neo) as a selectable marker were engineered, and the effectiveness of these vectors for expression of the reporter gene was evaluated after transfection into the packaging cell line GP+E86. It was found that the highest levels of reporter gene expression were attained with constructs ordered 5′ long terminal repeat (LTR)-TAC-internal promoterneo-3′ LTR. In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli β-galactosidase gene (lacZ) instead of TAC, because a very sensitive detection method was available for lacZ. For transduction of hematopoietic progenitors, packaging cell lines producing recombinant viruses were cultured in a Transwell hung into a Dexter-type bone marrow (BM) culture. The BM cells were selected with G418, and transferred into irradiated recipient mice. LacZ enzyme activity was detectable in the peripheral blood lymphocytes (PBL) of recipients taken 8 wk after reconstitution.     

Loss of cryptochrome reduces cancer risk in p53 mutant mice

It is commonly thought that disruption of the circadian clock increases the cancer incidence in humans and mice. However, it was found that disruption of the clock by the Cryptochrome (Cry) mutation in mice did not increase cancer rate in the mutant mice even after exposing the animals to ionizing radiation. Therefore, in this study we tested the effect of the Cry mutation on carcinogenesis in a mouse strain prone to cancer because of a p53 mutation, with the expectation that clock disruption in this sensitized background would further increase cancer risk. Paradoxically, we find that the Cry mutation protects p53 mutant mice from the early onset of cancer and extends their median lifespan ≈50%, in part by sensitizing p53 mutant cells to apoptosis in response to genotoxic stress. These results suggest alternative therapeutic approaches in management of cancers associated with a p53 mutation.     

Protection of hematopoietic cells from O6-alkylation damage by O6-methylguanine DNA methyltransferase gene transfer: studies with different O6-alkylating agents and retroviral backbones

Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.     

缺锌对衰老小鼠抗氧化系统和肝脏DNA损伤修复功能的影响

目的　通过D 半乳糖诱导小鼠衰老模型 ,探讨缺锌对衰老小鼠抗氧化系统和肝脏DNA损伤与修复的影响。　方法　雄性 3月龄小鼠 70只 ,随机分成 5组 :青年组、衰老模型组、衰老缺锌组、衰老配喂组和衰老补锌组。各衰老组按 10 0mg /kg经颈背部皮下给予D 半乳糖注射液 ,青年组给予等剂量的生理盐水 ,连续 30d。衰老缺锌组和补锌组喂饲缺锌饲料 (含锌 1 6 1μg/kg) ,其他组喂饲正常锌饲料 (含锌 5 0 μg/kg) ,最后 2周补锌组喂饲补锌饲料 (10 0 μg/kg)。第 30天处死小鼠 ,取样检测血清锌、肝锌、超氧化物歧化酶、丙二醇、肝脂褐质和DNA损伤情况。　结果　与衰老模型组相比 ,衰老缺锌组血清锌 (0 5 3± 0 1)mg/L、肝锌 (14 5 4± 2 18)mg/L水平下降 ,血清和肝超氧化物歧化酶活性〔(14 2 87± 10 16 )NU/ml和 (180 11± 13 2 2 )NU/ml,P <0 0 5〕降低 ,丙二醇含量升高 ,肝脂褐质含量增高 ;彗星试验显示衰老缺锌组小鼠肝DNA损伤加重 ,彗星细胞尾长 /总长比值显著增加。补锌后上述指标均有改善。　结论　锌可有效的影响衰老的速度和程度 ,缺锌可加速衰老的进程 ,适当补锌有助于延缓衰老。     

骨性关节炎软骨修复与基因治疗

软骨修复是目前治疗骨关节炎最有前景的方法。间充质干细胞体外定向培养成软骨细胞解决了软骨细胞难以获得的问题。不同细胞因子在软骨细胞分化过程中可发挥不同作用。通过对其多靶点干预，促进软骨细胞分化、生长和软骨修复，可达到治疗骨关节炎的目的。用间充质干细胞进行骨关节炎软骨缺损的基因治疗是一种新的治疗手段。     

DNA polymerase ε and δ proofreading suppress discrete mutator and cancer phenotypes in mice

Organisms require faithful DNA replication to avoid deleterious mutations. In yeast, replicative leading- and lagging-strand DNA polymerases (Pols ε and δ, respectively) have intrinsic proofreading exonucleases that cooperate with each other and mismatch repair to limit spontaneous mutation to less than 1 per genome per cell division. The relationship of these pathways in mammals and their functions in vivo are unknown. Here we show that mouse Pol ε and δ proofreading suppress discrete mutator and cancer phenotypes. We found that inactivation of Pol ε proofreading elevates base-substitution mutations and accelerates a unique spectrum of spontaneous cancers; the types of tumors are entirely different from those triggered by loss of Pol δ proofreading. Intercrosses of Pol ε-, Pol δ-, and mismatch repair-mutant mice show that Pol ε and δ proofreading act in parallel pathways to prevent spontaneous mutation and cancer. These findings distinguish Pol ε and δ functions in vivo and reveal tissue-specific requirements for DNA replication fidelity.     

Metronidazole-induced hepatotoxicity in a patient with xeroderma pigmentosum: A case report

Rationale:Whereas metronidazole-induced hepatotoxicity is quite rare in the general population, in individuals carrying a nucleotide excision repair disorder, namely Cockayne syndrome, there is a high risk of developing this complication.Patient concerns:We report the case of a 44-year-old man, affected by xeroderma pigmentosum, who was admitted to the hospital presenting aspiration pneumoniae caused by worsening dysphagia and with severe hepatotoxicity during the hospitalization.Diagnoses:Acute hepatitis, which was leading to acute liver failure, occurred during antibiotic treatment with metronidazole and ceftazidime with an elevation of liver enzymes consistent with hepatocellular damage pattern.Interventions:Hydration with glucose 5% solution, pantoprazole and vitamin K were administered, meanwhile other causes of hepatitis were ruled out and the ongoing antibiotic treatment was stopped suspecting a drug-induced liver injury.Outcomes:Liver function nearly completely recovered 1 month later with a first rapid improvement, within few days, of aminotransferases and coagulation studies, and slower of cholestatic enzymes.Lessons:We describe the first case available in the literature of hepatotoxicity associated with metronidazole treatment in a xeroderma pigmentosum patient. Clinicians therefore, based on this report and according to the possible underlying mechanism shared by other genetic diseases characterized by alterations in the pathway of DNA-repair, should consider such adverse event also in patients affected by this rare disease.     

GM-CSF Gene therapy using adenoviral vector in hamster pancreatic cancer

The aim of this study was to examine the antitumor effect of irradiated granulocyte macrophage-colony-stimulating factor (GM-CSF)-gene-transduced hamster pancreatic cancer cells and its relationship to the amount of GM-CSF produced by transduced tumor cells. Hamster pancreatic adenocarcinoma cells, HPD1NR, which spontaneously secrete 15.0 ± 0.4 pg/10⁶ cells per 24 h of GM-CSF, and HPD2NR cells, which do not secrete GM-CSF, were used. When these cells were infected with recombinant adenovirus harboring theGM-CSF gene, HPD1NR and HPD2NR secreted 624.2 ± 9.9 and 157.8 ± 5.7 pg/10⁶ cells per 24 h, respectively. Vaccination with irradiatedGM-CSF-secreting HPD2NR completely protected syngeneic hamsters challenged with live parental cells. On the other hand, vaccination with irradiated HPD1NR protected 60% of hamsters from tumor development after challenge with parental cells. None of the tumor-free hamsters initially vaccinated with irradiated GM-CSF-producing HPD2NR cells developed tumor upon repeated challenge with parental cells during the entire observation period. IrradiatedGM-CSF-gene-transduced hamster pancreatic cells are promising as a novel adjuvant cancer therapy after surgery for primary and metastatic pancreatic cancer. The results indicate the necessity for a therapeutic strategy for cancer based on the cytokine status of tumors.