The cyclin-dependent kinase inhibitor p21CDKN1A as a target of anti-cancer drugs.

p21CDKN1A (WAF1/CIP1/SDI1), the cyclin-dependent kinase (CDK) inhibitor belonging to the Cip/Kip family, was first described as a potent inhibitor of cell proliferation and DNA replication, both in physiological conditions and after DNA damage. More recently, p21 has been recognized to play additional and fundamental roles in other important pathways, including regulation of transcription, apoptosis and DNA repair. Knock-out mouse studies combined with biochemical and functional analysis of cells in culture have indicated a tumor suppressor activity for p21. However, these lines of evidence have been complicated by other findings indicating that p21 can exhibit oncogenic properties. In fact, the evidence that p21 expression may lead to proliferation arrest, is counterbalanced by the rescue of tumor cells from drug-induced apoptosis, and by promoting a metastatic potential. For these reasons, p21 is considered a protein with a dual behavior, with potential benefits, as well as dangerous effects of its expression in malignant cells. Thus, the effectiveness of targeting p21 expression for antitumor therapy needs to be carefully evaluated accordingly. This review summarizes the functions and regulations of p21, and focuses on its involvement in human diseases (particularly cancer), and on the pharmacological approaches to target p21 expression (either positively or negatively) for anticancer therapy. Based on these approaches, the search for new molecules that are able to promote the tumor-suppressor activity, and/or to interfere with the oncogenic properties of p21, could be promising.

The resveratrol analogue 4,4'-dihydroxy-trans-stilbene inhibits cell proliferation with higher efficiency but different mechanism from resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antiproliferative activity. We previously found that 4-hydroxy group in the trans conformation was absolutely required for the inhibition of cell proliferation. In the present work we have synthesized the resveratrol analogue 4,4'-dihydroxy-trans-stilbene, which contains two OH in 4' and 4 positions, with the aim of developing a compound with an antiproliferative potential higher than that of resveratrol, on the basis of the correlation between structure and activity previously observed. In comparison with resveratrol, 4,4'-dihydroxy-trans-stilbene inhibited cell clonogenic efficiency of fibroblasts nine times more although with a different mechanism. First, 4,4'-dihydroxy-trans-stilbene induced predominantly an accumulation of cells in G1 phase, whereas resveratrol perturbed the G1/S phase transition. Second, although both compounds were able to inhibit DNA polymerase (pol) delta in an in vitro assay, 4, 4'-dihydroxy-trans-stilbene did not affect pol alpha activity. Finally, 4,4'-dihydroxy-trans-stilbene increased p21(CDKN1A) and p53 protein levels, whereas resveratrol led to phosphorylation of the S-phase checkpoint protein Chk1. Taken together, our results demonstrated for the first time that the two hydroxyl groups on 4- and 4'- positions of the stilbenic backbone enhance the antiproliferative effect and introduce additional targets in the mechanism of action of resveratrol. In conclusion, 4,4'-dihydroxy-trans-stilbene has potent antiproliferative activities that differ from the effect of resveratrol shown in this system, suggesting that it warrants further development as a potential chemopreventive or therapeutic agent.

Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells.

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.

Flavonoids uptake and their effect on cell cycle of human colon adenocarcinoma cells (Caco2).

Green tea, mainly through its constituents epigallocatechin gallate, epigallocatechin, epicatechin gallate and epicatechin, has demonstrated anticarcinogenic activity in several animal models, including those for skin, lung and gastro-intestinal tract cancer, although less is known about colorectal cancer. Quercetin, the major flavonoid present in vegetables and fruit, exerts potential anticarcinogenic effects in animal models and cell cultures, but less is known about quercetin glucosides. The objectives of this study were to investigate (i) the antioxidant activity of the phenolic compounds epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside; (ii) the cytotoxicity of different concentrations of epicatechin, epigallocatechin gallate, and gallic acid; (iii) the cellular uptake of epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside and (iv) their effect on the cell cycle. Human colon adenocarcinoma cells were used as experimental model. The results of this study indicate that all dietary flavonoids studied (epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside) show a significant antioxidant effect in a chemical model system, but only epigallocatechin gallate or gallic acid are able to interfere with the cell cycle in Caco2 cell lines. These data suggest that the antioxidant activity of flavonoids is not related to the inhibition of cellular growth. From a structural point of view, the galloyl moiety appears to be required for both the antioxidant and the antiproliferative effects.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes.

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antioxidant and antiproliferative activities. In this study we have investigated whether these properties are dependent on similar or different structural determinants of the molecule. To this purpose, resveratrol derivatives, in which all or each single hydroxylic function were selectively substituted with methyl groups, were synthesized. Analogues with the stilbenic double bond reduced or with the stereoisometry modified were also investigated. The antioxidant activity of these compounds was evaluated by measuring the inhibition of citronellal thermo-oxidation, or the reduction of 2,2-diphenyl-1-picrylhydrazyl radical. In addition, the protection against lipid peroxidation was determined in rat liver microsomes, and in human primary cell cultures. The antiproliferative activity was evaluated by a clonogenic assay, and by analysis of cell cycle progression and DNA synthesis. The results showed that the hydroxyl group in 4' position is not the sole determinant for antioxidant activity. In contrast, the presence of 4'-OH together with stereoisometry in the trans-conformation (4'-hydroxystyryl moiety) was absolutely required for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of DNA synthesis was induced by a direct interaction of resveratrol with DNA polymerases alpha and delta.

p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites.

The cyclin-dependent kinase inhibitor p21(waf1/cip1) is known to impair DNA synthesis by binding to PCNA, the co-factor of DNA polymerases delta and epsilon. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA repair efficiency were investigated in p21-null human fibroblasts obtained by targeted homologous recombination. After UV-C irradiation, p21-/- cells showed a threefold reduction in clonogenic survival and an increased susceptibility to apoptosis, as compared with parental p21+/+ cells. Removal of cyclobutane pyrimidine dimers was significantly reduced in p21-/- cells both in the whole genome, and at the level of the rDNA gene cluster, as determined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repair sites in p21-/- fibroblasts, was comparable to that observed in parental p21+/+ cells. However, PCNA remained associated with DNA for a longer period in p21-/- than in p21+/+ cells. These results suggest that in human cells, p21 is required for NER at a step located downstream the recruitment of PCNA to DNA repair sites.

The antiproliferative effect of beta-carotene requires p21waf1/cip1 in normal human fibroblasts.

In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.

Zidovudine-induced experimental myopathy: dual mechanism of mitochondrial damage.

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers. In the same animal models, the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a partial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis. In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy.

The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity.

The specificity and the temporal location of cell cycle arrest induced by the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investigated in normal human fibroblasts. Effects on the cell cycle were compared with those induced by the kinase inhibitor staurosporine, which arrests normal cells in early G1 phase by acting upstream of CDK2. Consistent with their in vitro activity, olomoucine and roscovitine, but not the related compound iso-olomoucine, induced a dose-dependent arrest in G1 phase. Following removal of CDK inhibitors, cells resumed cycle progression entering S phase with a kinetics faster than staurosporine-treated samples. Cellular levels of PCNA, cyclin D1, and cyclin E were not affected by the CDK inhibitors. In contrast, staurosporine significantly reduced the levels of these proteins, as determined by immunocytometry and Western blot analysis. Cyclin A was detectable only in some cells remaining in the G2 + M compartment of samples treated with CDK inhibitors, but not in samples treated with staurosporine. Significant reduction in the hyperphosphorylated forms of retinoblastoma protein was found in samples treated with CDK inhibitors, while only hypophosphorylated forms were observed in staurosporine-treated samples. Concomitantly, CDK2, but not CDK4, activity immunoprecipitated from cells treated with olomoucine or roscovitine was markedly inhibited. These results suggest that in normal cells, CDK2 kinase activity is the specific target of olomoucine and roscovitine.

Involvement of the proliferating cell nuclear antigen (PCNA) in DNA repair induced by alkylating agents and oxidative damage in human fibroblasts.

The involvement of the proliferating cell nuclear antigen (PCNA) in the process of DNA repair induced by alkylating agents or by oxidative damage was investigated in human quiescent fibroblasts by immunofluorescence and flow cytometry. Transition from soluble to the DNA-bound form of PCNA, was taken as the parameter to determine its involvement in repair DNA synthesis. Treatment with the alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine resulted in the rapid and dose-dependent increase in the nuclear binding of PCNA. Similar results were obtained with compounds such as hydrogen peroxide or tert-butyl hydroperoxide, which are known to induce oxidative DNA damage. Tert-butyl hydroperoxide may also generate malondialdehyde through a reaction of lipid peroxidation. This mutagenic and carcinogenic product has been previously shown to form adducts with DNA. Therefore, the possibility that tert-butyl hydroperoxide could induce DNA damage through this pathway was investigated by incubating cells directly in the presence of malondialdehyde. Such treatment resulted in an increase in immunofluorescence associated with nuclear-bound PCNA. The ability of oxidative and alkylating agents to induce the nuclear binding of PCNA was also assessed in proliferating cells. In these conditions, treatment with hydrogen peroxide or methylmethane sulfonate, resulted in an increase in nuclear-bound PCNA in the G1 and in the G2 + M compartments, but not in S phase. At longer times after treatment, PCNA immunostaining was reduced to basal levels, while an increase in nuclear binding of p21(waf1/cip1) protein was found in concomitance with cell-cycle arrest. These results indicate that agents inducing DNA base alterations in vivo, promote the nuclear binding of PCNA. These lines of evidence support the role of a PCNA-dependent reaction in the base excision repair system.

Nuclear association of cyclin D1 in human fibroblasts: tight binding to nuclear structures and modulation by protein kinase inhibitors.

The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts by using hypotonic detergent extraction procedures, immunofluorescence quantitation with flow cytometry, and Western blot analysis. About 20% of the total cellular levels of cyclin D1 was found to be tightly bound to nuclear structures, being the complex formation resistant to DNase I treatment and to high salt extraction. Maximal levels of the insoluble form of the protein were found in the middle to late G1 phase of the cell cycle. Cell fractionation and immunoprecipitation techniques after in vivo 32P-labeling showed that both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions were reactive to an anti-phosphotyrosine antibody, while only the latter was detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which induces a cell cycle arrest in early G1 phase, strongly reduced cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cyclin D1 levels was reduced by about 60%, compared with the control value. The protein kinase A specific inhibitor isoquinoline-sulfonamide (H-89) induced a similar reduction in the ratio, with no significant modification in the total amount of protein. In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, consistently increased by 30-50% the ratio of nuclear-bound/total amount of the cyclin protein. These results suggest that, during the G1 phase, formation of an insoluble complex of cyclin D1 occurs at nuclear matrix structures and that this association is mediated by a protein kinase A-dependent pathway.

Effect of beta-carotene on cell cycle progression of human fibroblasts.

The uptake of beta-carotene (BC) and its effect on the cell cycle progression of normal human fibroblasts in primary culture were investigated by using two different delivery methods: exposure to BC solubilized in the organic solvent tetrahydrofuran (THF) or to BC incorporated into dipalmitoylphosphatidylcholine (DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescence detection and flow cytometric analysis of the proliferating cell nuclear antigen (PCNA). In contrast to THF, which induced a marked reduction in the number of cells in S phase and in the extent of PCNA immunolabeling, DPPC liposomes proved to be an effective delivery system that does not interfere with cell proliferation. Cellular uptake of 0.23 nmol/10(6) cells was found after 24 h incubation in BC-containing DPPC liposomes. This value increased to 1.2 nmol/10(6) cells after 72 h. After the first day of incubation, the number of cells in S phase was reduced by approximately 50%, with a consequent accumulation of cells in G1 phase. This effect was maintained up to 3 days incubation, with no detectable effects on cell viability. This cell cycle delay was found to be reversible, returning the percentage of cells in S phase to the control value 24 h after removal of BC from the medium. In order to determine whether the activity of BC could be attributed to the molecule itself or to its conversion into retinoids, the production of BC metabolites was assessed. Analysis of cellular levels of retinoids failed to demonstrate the presence of retinal, retinol, retinoic acid or retinyl esters during an incubation period of 6 days. These results suggest that in normal human fibroblasts, BC induces a cell cycle delay in the G1 phase and that this effect is independent of conversion to known retinoids.

p21waf1/cip1 protein associates with the detergent-insoluble form of PCNA concomitantly with disassembly of PCNA at nucleotide excision repair sites.

Evidence is presented that after exposure of normal human fibroblasts to UV-C light, nuclear binding of the proliferating cell nuclear antigen (PCNA) required for nucleotide excision repair, was rapidly triggered in the G1 and G2 phases of the cell cycle. Association to repair sites of the detergent-insoluble form of PCNA reached a peak 15-30 min after irradiation, and then decreased to basal levels within 24-48 h. In contrast, the nuclear association of p21 protein showed a slower kinetics, reaching maximal levels between 24 and 48 h but, similarly to PCNA, occurring only in G1 and G2 phases. Although the two proteins are known to be associated as detergent-soluble proteins, it is unknown whether they associate also in the detergent-insoluble form. To address this question, the chromatin-bound form of PCNA was released by using DNAse I. DNA digestion resulted in the almost complete release of PCNA from its binding sites, while only about 60% of nuclear-bound p21 could be solubilized. Immunoprecipitation of PCNA and p21 released by enzymatic digestion showed that p21 was associated with PCNA bound to late DNA repair sites. These results indicate that during nucleotide excision repair, nuclear binding of PCNA precedes that of p21 protein, and suggest that temporal association of p21 with the detergent-insoluble fraction is coincident with the disassembly of PCNA from DNA repair sites.

Effects of beta-carotene and alpha-tocopherol on photogenotoxicity induced by 8-methoxypsoralen: the role of oxygen.

The protective effect of beta-carotene (beta-C) and alpha-tocopherol (alpha-T), singularly and in equimolar mixtures, toward the photomutagenicity induced by 8-methoxypsoralen (8-MOP), at different oxygen partial pressure (pO2), was evaluated in two different experimental models: Salmonella typhimurium TA102 and Saccharomyces cerevisiae D7. After phototreatment with 8-MOP, the results show a lethal effect under hypoxic conditions in both experimental model systems, an increase in revertants associated to the pO2 increase in S. typhimurium TA102, and a decrease in revertants and convertants associated to the pO2 increase in S. cerevisiae D7. In S. typhimurium TA102, in atmospheric condition, beta-C and alpha-T (1.86 or 18.6 microM) show a protective effect only at the higher dosage. Alpha-T was more protective than beta-C. The equimolar mixtures show an antimutagenic effect at both dosage used with a synergistic effect at lower dosage and an additive antimutagenic activity at higher dosage. An inhibition of the spontaneous mutagenicity by mixtures at higher dosage was also observed. The results obtained in S. typhimurium TA102 show an antimutagenic effects of beta-C, alpha-T and their mixture at 190 mmHg pO2, confirming the data obtained in air condition. At 380 mmHg pO2, alpha-T and the mixture show a significant antimutagenic activity; at 570 mmHg pO2, only alpha-T is protective. At 760 mmHg pO2, no protective effect was observed by the two antioxidants, and beta-C increases the photomutagenicity induced by 8-MOP. In S. cerevisiae D7 a protective effect was only observed at 380 mmHg pO2 with the mixture. No antigenotoxic effect was found in the other experimental conditions, even if the uptake of the two antioxidants was confirmed by HPLC. Our results underline the role of oxygen in the photomutagenicity induced by 8-MOP and in the antimutagenic activity of beta-C and alpha-T. This is the first report confirming in a cellular experimental model the data obtained in some chemical systems: the protective effect of beta-C only at low pO2 and the synergistic effect of mixture of beta-C and alpha-T.

Photoinduction of micronuclei by 4,4',6-trimethylangelicin and 8-methoxypsoralen in different experimental models.

The frequencies of micronuclei induced by treatment with 4,4',6-trimethylangelicin (TMA) and 8-methoxypsoralen (8-MOP) have been compared in the following experimental models: (1) peripheral normochromatic erythrocytes (NCE) during 10 days after single p.o. administration of TMA or 8-MOP in male and female mice; (2) peripheral NCE during photocarcinogenesis by TMA or 8-MOP topically administered to female mice; (3) primary cultures of human skin fibroblasts treated with TMA or 8-MOP. The frequency of micronuclei in peripheral NCE of mice (both sexes) was significantly enhanced after p.o. administration of TMA or 8-MOP. This latter was more active than TMA in inducing chromosomal damage. No increased frequencies of micronuclei in peripheral NCE were detected in mice subjected to TMA or 8-MOP photocarcinogenic treatment, even when malignancies developed. In human fibroblast cultures, at equimolar concentrations, the induction of lethal effects by TMA in the presence of 365-nm radiation was higher than that exerted by 8-MOP. At equal survival, however, TMA showed practically the same activity as 8-MOP in the induction of micronuclei. Our findings provide evidence of genotoxicity by TMA administered p.o. without irradiation and give further information about photogenotoxicity of these substances.

Nuclear binding of cell cycle-related proteins: cyclin A versus proliferating cell nuclear antigen (PCNA).

We have investigated the cell cycle-dependent nuclear binding of cyclin A and of the proliferating cell nuclear antigen (PCNA) in asynchronously growing human fibroblasts. To this purpose, we have applied flow cytometry immunofluorescence, a powerful technique for elucidating the cell cycle phase during which the nuclear binding occurs. We have observed that, in striking contrast with the distribution of nuclear-bound PCNA which is restricted to S phase, the immunofluorescence signal of the nuclear-bound form of cyclin A is high in the G1 and G2 phases of the cell cycle. These results suggest the involvement of nuclear-bound cyclin A in the G1/S and G2/M phase transitions.

Proliferating cell nuclear antigen bound to DNA synthesis sites: phosphorylation and association with cyclin D1 and cyclin A.

Evidence is presented that association of proliferating cell nuclear antigen (PCNA) with nuclear chromatin in human fibroblasts is related to the phosphorylation status of the protein. Using a hypotonic lysis procedure to extract the soluble form of PCNA, it has been shown that the remaining nuclear-bound form, predominantly in S-phase cells, is highly phosphorylated. Cells in early G1, or in G2 + M phases, contain basal levels of the bound form of the protein that is only weakly phosphorylated. Using fractionated immunoprecipitation techniques, PCNA was found to be associated with cyclin A in both soluble and insoluble fractions. In contrast, association of PCNA with cyclin D1 was found in the soluble fraction, while no detectable levels were present in the insoluble fraction. Immunofluorescence labeling and flow cytometric analysis of the cell cycle distribution of cyclin D1 and cyclin A showed that, like PCNA, maximal levels of both proteins were bound to nuclear structures at the G1/S phase boundary. These results suggest that binding of PCNA to DNA synthesis sites occurs after phosphorylation. Association with cyclin D1 and cyclin A might occur in a macromolecular complex assembled at the G1/S phase boundary to drive activation of DNA replication factors.

Genotoxicity assay of five pesticides and their mixtures in Saccharomyces cerevisiae D7.

Four organophosphorus pesticides (azinphos-methyl, diazinone, dimethoate, and pirimiphos-methyl), and one carbamate (benomyl) were tested for cytotoxicity, reverse mutation and gene conversion in Saccharomyces cerevisiae D7, with and without the S9 metabolic system. Furthermore, two mixtures of the above compounds, namely benomyl + pirimiphos-methyl (6/1 ratio) and dimethoate + diazinone + azinphos-methyl (10/4/6 ratio) were tested in the same experimental model. Azinphos-methyl, benomyl, and pirimiphos-methyl alone did not induce any genotoxic effect, whereas azinphos-methyl and diazinone were active in inducing reversion and gene conversion. The benomyl + pirimiphos-methyl mixture did not show any genotoxic activity. The dimethoate + diazinone + azimphos-methyl mixture was genotoxic, although an antagonistic effect between the components was observed. The addition of S9 post-mitochondrial liver fraction decreased the activity of both single and mixed genotoxic agents.