Silvano Nocentini

Photochemotherapy using pyridopsoralens

Aiming to decrease the acute side effects and genotoxic hazards of PUVA, pyrido (3,4-C) psoralen (PP) and 7-methyl pyrido (3,4-C) psoralen (MPP) were synthesized and studied. Their UVA maximum absorption lies at 325 and 330 nm, respectively. Their photostability is comparable to that of 8-MOP. They complex to DNA in the dark, and, in the presence of UVA, produce only monoadditions to DNA, as shown by fluorescence and DNA denaturation-renaturation studies. In diploid eukaryotic yeast they are more effective than 8-MOP for the induction of lethal effects and mitochondrial damage. Their mutagenic activity per unit dose of UVA is in the same range as that of 8-MOP. However, per viable cell they are clearly less mutagenic than 8-MOP. This difference is also observed for recombinogenic activity. No oxygen effect is observed. In mammalian cells the following ranges of effectiveness are found: inhibition of DNA synthesis in human fibroblasts: MPP greater than PP greater than 8-MOP; mutagenic activity in V79 Chinese hamster cells: MPP greater than PP greater than 8-MOP; cell transforming ability in C3H embryonic mouse cells: MPP greater than 8-MOP greater than PP as a function of UVA dose, and: 8-MOP greater than MPP greater than PP as a function of survival; induction of sister chromatic exchanges (SCE) per unit dose: MPP greater than PP greater than 8-MOP in the linear part of the induction curve, and : 8-MOP greater than PP greater than MPP at the maximum level of SCE obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-β1 enhances the lethal effects of DNA-damaging agents in a human lung-cancer cell line

In tissue culture conditions, exogeneous active transforming growth factor‐β1 (TGF‐β1) enhances the lethal effect of DNA‐damaging agents (UV‐C, gamma rays, cisplatin, methotrexate and 5‐fluorouracil) toward human A549 cells and mink Mv1Lu cells, as detected by the loss of their capacity to give rise to colonies; both these cell lines harbor a wild‐type p53, as determined by immunoprecipitation. Contrastingly, the sole effect of the cytokine used alone is to inhibit reversibly the multiplication of the same cells without further impairing, once withdrawn from their environment, their capacity to divide and give rise to colonies. The lethal synergy between TGF‐β1 and UV‐C was studied on mink and human cell lines, and the biomodulation by TGF‐β1 of cell killing by cisplatin, gamma rays, 5‐fluorouracil or methotrexate was tested only on human cells. As investigated with UV‐C‐irradiated human A549 cells, TGF‐β1 appears to enhance apoptosis rather than to disturb the repair of DNA photolesions (mainly pyrimidine dimers) by the nucleotidic excision repair pathway according to results of nucleosomal ladder and comet tests. Our data raise the possibility that, in vivo, TGF‐β1 might affect the curative and/or undesirable secondary side effects of cancer therapy. Int. J. Cancer 72:356–361, 1997.

Exacerbating Effect of Vitamin E Supplementation on DNA Damage Induced in Cultured Human Normal Fibroblasts by UVA Radiation

Abstract The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320–380 nm) (UVA). Cells were incubated in medium containing α-tocopherol, α-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2·−) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2·− reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.

Induction of cytogenetic effects in human fibroblast cultures after exposure to formaldehyde or X-rays.

Cytogenetic changes induced by formaldehyde (FA) in exponentially growing human fibroblasts were compared with those produced by X-rays. Chromosome aberrations of different types were detected. Exposure to 2 mM FA for 15 min resulted, quantitatively and qualitatively, in effects comparable to those produced by an X-ray dose of 100 rad. X-Rays at higher exposures produced types of chromosome aberrations that differed from those induced by higher exposures to FA.

Mitochondrial Alterations in Fanconi Anemia Fibroblasts Following Ultraviolet A or Psoralen Photoactivation

Abstract The genetic disease Fanconi anemia (FA), generally considered to be a DNA repair defect, has also been related to a deficiency in cellular defense against reactive oxygen species (ROS). Results show that mitochondrial matrix densification occurs rapidly and transiently in FA fibroblasts following 8-methoxypsoralen (8-MOP) photoreaction or ultraviolet A (320 to 380 nm) (UVA) irradiation. This effect is oxygen dependent because it is more important under 20 than under 5% oxygen tension. In contrast, in normal fibroblasts very little, if any, densification of mitochondrial matrix is induced by treatments even at the highest oxygen tension. The changes in matrix density in FA cells are accompanied by some modifications in transmembrane potential, linked to a Fenton-like reaction, and in mitochondrial cardiolipin content, differing from the responses of normal cells. These data are indicative of some sort of membrane damage induced by 8-MOP photoreaction and UVA irradiation, to which FA cells appear to be particularly sensitive.

Apoptotic response of malignant rhabdoid tumor cells

BackgroundMalignant rhabdoid tumors (MRTs) are extremely aggressive and resist current radio- and chemotherapic treatments. To gain insight into the dysfunctions of MRT cells, the apoptotic response of a model cell line, MON, was analyzed after exposure to several genotoxic and non-genotoxic agents employed separately or in association.ResultsFluorescence microscopy of chromatin morphology and electrophoretic analysis of internucleosomal DNA fragmentation revealed that MON cells were, comparatively to HeLa cells, resistant to apoptosis after treatment with etoposide, cisplatin (CisPt) or X-rays, but underwent some degree of apoptosis after ultraviolet (UV) C irradiation. Concomitant treatment of MON cells with X-rays or vinblastine and the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin resulted in synergistic induction of apoptosis. Western blot analysis showed that the p53 protein was upregulated in MON cells after exposure to all the different agents tested, singly or in combination. In treated cells, the p53 downstream effectors p21WAF1/CIP1, Mdm2 and Bax were induced with some inconsistency with regard to the accumulation of p53. Poly ADP-ribose polymerase (PARP) cleavage, indicative of ongoing apoptosis, occurred in UVC-irradiated cells and, especially, in cells treated with combinations of X-rays or vinblastine with wortmannin. However, there was moderate or no PARP cleavage in cells treated with CisPt, X-rays, vinblastine or wortmannin singly or with the combinations X-rays plus CisPt or vinblastine and CisPt plus vinblastine or wortmannin. The synergistic effect on the induction of apoptosis exerted by some agent combinations corresponded with synergy in respect of MON cell growth inhibition.ConclusionThese results suggest abnormalities in the p53 pathway and apoptosis control in MRT cells. The Ras/PI3-K/AKT signaling pathway might also be deregulated in these cells by generating an excess of survival factors. These dysfunctions might contribute to the resistance of MRTs to current antineoplastic treatments and could warrant consideration in the search of new therapeutic approaches.

3‐CARBETHOXYPYRANOCOUMARIN, A PHOTOREACTIVE DERIVATIVE OF XANTHYLETIN WITH INTERESTING PHOTOBIOLOGICAL PROPERTIES

Abstract— The photobiological activity of the newly synthesized pyranocoumarin derivative 3‐carbethoxypyranocoumarin, so‐called 3‐carbethoxyhomopsoralen (3‐CHPs) was studied in comparison to the known bifunctional furocoumarin 8‐methoxypsoralen {8‐MOP) and to the monofunctional furocoumarin 3‐carbethoxypsoralen (3‐CPs) in the presence of 365 nm irradiation using two eukaryotic cell systems, the yeast Saccharomyces cerevisiae and cultured normal human skin fibroblasts. 3‐Carbethoxyhomopsoralen is shown to be a photobiologically active compound capable of effectively photoinducing cytoplasmic “petite” mutants (mitochondrial damage), nuclear reversions and mitotic gene conversion in the diploid yeast strain D7. Per unit dose it is more effective than 8‐MOP and 3‐CPs for the induction of cytoplasmic “petite” mutants but less effective than 8‐MOP for the induction of nuclear reversions and mitotic gene conversion. A very moderate effect on cell survival is accompanied by a relatively strong genetic activity per viable cell. In human fibroblasts 3‐CHPs produces a stronger inhibition of DNA synthesis than 8‐MOP and 3‐CPs at low doses of 365 nm radiation. During post‐treatment incubation human fibroblasts recovered more easily from DNA synthesis and growth inhibitions photoinduced by 3‐CHPs than from those photoinduced by 8‐MOP. The results are in accord with the notion that 3‐CHPs is a highly photoreactive monofunctional compound inducing easily repairable lesions with a low lethal but significant mutagenic potential.

DNA ligase activity in carcinogen-treated human fibroblasts.

In an enzymological approach to study DNA repair mechanisms induced by carcinogen-treatment of mammalian cells, we have investigated how DNA ligase activity is affected by the treatment with several compounds producing different DNA lesions. Stationary cultures of human fibroblasts were exposed to various doses of carcinogens (UV-light at 254 nm, N-acetoxy-acetyl-aminofluorene, ethyl-methane sulfonate, N-methylnitro-nitrosoguanidine, mitomycin C and 4-nitroquinoline-N-oxide) at different time-intervals before preparing crude cellular extracts and assaying for ligase activity. Results have shown that: 1. UV-irradiation, AAAF, 4NQO or MMC treatment of cells induces a two-fold increase in the ligase activity compared to control cells within 48 hours following the treatment. 2. A partial purification of the enzyme from these cellular crude extracts by sedimentation through sucrose gradients has shown: a. DNA ligase activity from control cells presents a profile composed of two distinct peaks sedimenting respectively at about 4S and 7S; b. the carcinogen treatment of either repair-proficient human fibroblasts or repair-deficient xeroderma pigmentosum cells (complementation group A) seems to induce a specific increase of the 4S-form of DNA ligase.

Immunological probing of induction and repair of 8-methoxypsoralen photoadducts in DNA from Fanconi anemia and normal human fibroblasts: Quantitative analysis by electron microscopy

A direct method of immuno-electron microscopy has been employed to simultaneously determine 8-methoxypsoralen (8-MOP) photoinduced monoadducts (MA) and interstrand cross-links (CL), their relative localization along the DNA molecule, and their removal. It has been applied to DNA from cultures of Fanconi anemia (FA) fibroblasts (complementation groups A and D), and of normal human fibroblasts, following treatment by 8-MOP and 365 nm radiation. The immuno-reaction with monoclonal antibody 8G1 was performed on DNA extracted from the cells just after photoreaction, or after a 24 h repair period, and then denatured. Furan-side MA and also a significant proportion of pyrone-side MA were very efficiently immuno-detected. Only 1-2% CL were IgG-labeled. This is why CL were directly visualized and quantified on denatured DNA from the same cellular samples as used for immuno-detection. Results demonstrate that FA group A cells are not only impaired in the incision of CL, but also of MA. The response of FA group D cells is intermediate between normal and FA group A cells.

Effects of aphidicolin on the recovery of ribosomal RNA synthesis and on the repair of potentially lethal damage in UV irradiated simian and human cells

Abstract Transcription analysis have been employed as probe for monitoring DNA damage and repair in order to elucidate whether or not aphidicolin, a specific inhibitor of DNA polymerase α, affects the repair of UV damage. Results show that aphidicolin inhibits in a concentration-dependent way the recovery of ribosomal RNA transcription in both human and simian cells, indicating a direct involvement of α polymerase in DNA repair. This conclusion was further supported by the inhibitory action of aphidicolin on the repair of potentially lethal damage in UV-irradiated monkey cells.