Efecto del tratamiento con ácido zoledrónico e hidroxocobalamina sobre la microarquitectura ósea en ratones con periodontitis y osteoporosis inducida: Estudio preclínico / Effect of zoledronic acid and hydroxocobalamin treatment on bone microarchitecture in mice with induced periodontitis and osteoporosis: preclinical study

Objetivo. Evaluar el efecto del tratamiento con ácido zoledrónico e hidroxocobalamina sobre la microarquitectura ósea alveolar en ratones con periodontitis y osteoporosis inducidas. Métodos. Diseño experimental en fase preclínica. Se incluyeron 16 ratones hembras a quienes se les indujo osteoporosis mediante la ovariectomía total y también se indujo la periodontitis por inflamación por ligadura de seda negra 5/0 en el segundo molar maxilar, todos los protocolos fueron sometidos durante anestesia general. Los ratones se distribuyeron en 4 grupos: control, tratamiento con ácido zoledrónico, tratamiento con hidroxocobalamina y tratamiento combinado. A las 16 semanas, se realizó la autanasia, se realizó la disección para la evaluación mediante microtomografía; determinando la densidad mineral ósea (BMD), el volumen de hueso (BV/TV), espesor trabecular (Tb. Th), número de trabéculas (Tb.N), separación trabecular (Tb.Sp); se realizó el análisis descriptivo y bivariado mediante ANOVA de 1 vía considerando un 95% de nivel de confianza. Resultados. El grupo que recibió tratamiento combinado de ácido zoledrónico e hidroxocobalamina presentó mayor densidad mineral ósea (DMO), mayor volumen óseo (BV/TV) y menor separación trabecular (Tb.Sp) en comparación con el grupo de control (p<0,05). Conclusiones. El tratamiento combinado de ácido zoledrónico e hidroxocobalamina mejora las características microarquitectónicas óseas en ratones con osteoporosis y periodontitis inducidas.

Objective. Evaluate the effect of zoledronic acid and hydroxocobalamin treatment on alveolar bone microarchitecture in mice with induced periodontitis and osteoporosis. Methods. Experimental design in preclinical phase. Sixteen female mice were included in which osteoporosis was induced by total ovariectomy and periodontitis was also induced by inflammation by 5/0 black silk ligation of the maxillary second molar, all protocols were performed under general anesthesia. The mice were distributed into 4 groups: control, treatment with zoledronic acid, treatment with hydroxocobalamin and combined treatment. At 16 weeks, euthanasia was performed, dissection was performed for evaluation by microtomography; determining bone mineral density (BMD), bone volume (BV/TV), trabecular thickness (Tb.Th), number of trabeculae (Tb.N), trabecular separation (Tb.Sp); descriptive and bivariate analysis was performed using 1-way ANOVA with a 95% confidence level. Results. The group that received combined treatment of zoledronic acid and hydroxocobalamin presented higher bone mineral density (BMD), higher bone volume (BV/TV) and lower trabecular separation (Tb.Sp) compared to the control group (p<0.05). Conclusions. Combined treatment with zoledronic acid and hydroxocobalamin improves bone microarchitectural features in mice with induced osteoporosis and periodontitis.

Influence of the Surface Energy of Different Brands of Polymethyl Methacrylate on the Adherence of Candida albicans: An In Vitro Study.

OBJECTIVE: The objective of this study was to evaluate the influence of the surface energy of different brands of polymethyl methacrylate (PMMA) on the adherence of Candida albicans ATCC 10231 in an in vitro study. MATERIALS AND METHODS: The study had an in vitro, longitudinal, and comparative experimental design. The following groups were made: (1) Vitacryl versus controls (water, dimethyl sulfoxide, glycerol, diethylene glycol, and formamide); (2) Triplex versus the same controls; (3) Vitacryl versus Triplex (surface energy); and (4) Vitacryl versus Triplex (adhesion per cell/field). Adhesion was measured in the area of each field magnified 10 × 10, and with an increase in magnification to 40 × 10, very dense colonies of 0.152 mm2 were observed. RESULTS: The surface energy of Vitacryl and Triplex was 40.3 ± 0.3 and 39.5 ± 0.3N/m, respectively, showing statistically significant differences (P < 0.001). On the contrary, in relation to the adhesion per cell/field of C. albicans, Vitacryl presented 15.7 ± 1.1, whereas Triplex had 16.7 ± 2.3, with no significant differences (P = 0.058). CONCLUSION: In relation to the adhesion per cell/field of C. albicans, there was no evidence of significant differences between Vitacryl and Triplex.

Conocimientos sobre cáncer bucal en pacientes odontológicos de la Universidad Nacional Mayor de San Marcos / Knowledge about oral cancer among dental patients from the National University of San Marcos

RESUMEN Introducción: El cáncer bucal es un problema de salud pública mundial, el cual constituye la sexta causa más común de muerte relacionada con el cáncer. El conocimiento y concientización de la población sobre esta enfermedad es importante para reducir su alta tasa de mortalidad. Objetivo: Determinar el nivel de conocimiento sobre cáncer bucal en pacientes adultos que acudieron a la Facultad de Odontología de la Universidad Nacional Mayor de San Marcos el año 2017. Métodos: Estudio observacional, descriptivo y transversal. La población estuvo compuesta por los pacientes que acudieron a la clínica de la Facultad de Odontología. La muestra fue de 223 pacientes y se obtuvo por un muestreo probabilístico aleatorio sistemático. El instrumento de evaluación fue una encuesta de 11 preguntas cerradas divididas en 5 dominios: conocimientos generales, factores de riesgo, signos y síntomas, repercusiones y prevención del cáncer bucal. El rango de calificación fue bajo, regular, alto. Resultados: El 56,5 por ciento (n= 126) de pacientes presentó un nivel de conocimiento bajo, 40,4 por ciento (n= 90) un nivel regular y 3,1 por ciento (n= 7) un nivel alto. El nivel de respuesta promedio fue 12,14 ± 2,90 (IC95 por ciento [10,17-14,89]). El sexo y la edad no tuvieron relación significativa con el nivel de conocimiento (p= 0,45 y p= 0,52, respectivamente); sin embargo, el nivel de educación sí tuvo relación significativa (p= 0,009). Conclusiones: En la población estudiada, el nivel de conocimiento sobre cáncer bucal es predominantemente bajo. El sexo y la edad no tienen influencia sobre el nivel de conocimiento de cáncer bucal, pero sí el nivel de educación(AU)

ABSTRACT Introduction: Oral cancer is a world health problem. It constitutes the sixth leading cause of cancer-related death. Knowledge about and awareness of this disease among the population is important to reduce its high mortality rate. Objective: Determine the level of knowledge about oral cancer among adult patients attending the Dental School at the National University of San Marcos in the year 2017. Methods: A cross-sectional observational descriptive study was conducted. The study population was the patients attending the Dental School clinic. The sample was 223 patients selected by systematic probabilistic random sampling. The evaluation tool was a survey containing 11 closed-ended questions divided into five domains: general knowledge, risk factors, signs and symptoms, repercussions and prevention of oral cancer. The grading scale was low, fair or high. Results: Of the patients surveyed, 56.5 percent (n= 126) had a low knowledge level, 40.4 percent (n= 90) a fair level and 3.1 percent (n= 7) a high level. Average answer level was 12.14 ± 2.90 (CI 95 percent [10.17-14.89]). Sex and age did not have a significant relationship to knowledge level (p= 0.45 and p= 0.52, respectively). However, educational level did have a significant relationship (p= 0.009). Conclusions: In the study population the level of knowledge about oral cancer is predominantly low. Sex and age do not have an influence on the level of knowledge about oral cancer, but educational level does(AU)

Signaling of DNA damage is not sufficient to induce p53 response: (re)activation of wt p53 protein strongly depends on cellular context.

It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS(3) cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS(3) cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS(3) cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS(3) cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.

A new, unexpected action of olomoucine, a CDK inhibitor, on normal human cells: up-regulation of CLIMP-63, a cytoskeleton-linking membrane protein.

Inhibition of cyclin-dependent kinases (CDKs) is a novel strategy in the therapy of human malignancies. The pharmacological CDK inhibitors representing a few distinct classes of compounds exert different target specificity. Considering the fact that dividing and quiescent cells differ in their CDK activity and in the pattern of their expression, one might expect that anti-proliferative efficiency of the pharmacological CDK inhibitors would depend on the mitotic index of treated cells. The present article shows that olomoucine (OLO), a weak CDK2 inhibitor has new, unexpected activity. At concentrations up to 100 microM OLO did not inhibit proliferation of normal human cells, but arrested growth of human HL-60 leukemia cells. The anti-proliferative effect of OLO was clearly weaker than that of roscovitine (ROSC). Surprisingly, OLO at low doses strongly up-regulated a cellular protein with approximately 65 kDa in normal, but not in immortalized and cancer cells. By mass spectrometric analysis CLIMP-63, a cytoskeleton-linking membrane protein was identified as the major component of the up-regulated protein band. These results were subsequently confirmed by immunoblotting. Further experiments revealed that OLO, but not ROSC, strongly up-regulates CLIMP-63 in a dose- and time-dependent manner solely in senescent cells.

Effect of distinct anticancer drugs on the phosphorylation of p53 protein at serine 46 in human MCF-7 breast cancer cells.

Roscovitine (ROSC), a potent cyclin-dependent kinase inhibitor (CDI), inactivates cyclin-dependent kinase (CDK)2 resulting in the arrest of human MCF-7 breast cancer cells in G2 phase of the cell cycle. We have recently observed a strong activation of wild-type (wt) p53 protein in human MCF-7 breast cancer cells upon treatment with ROSC implicating that upregulated p53 might additionally modulate the primary action of ROSC. ROSC stabilized wt p53 protein resulting in a marked extension of its half-life. Since ROSC exhibits low cytotoxicity, it seems to upregulate p53 protein in a way different from DNA damage. ROSC induced phosphorylation of p53 protein at serine 46. Therefore, we decided to examine whether other anticancer drugs are also able to induce phosphorylation of wt p53 protein at serine 46. Exposure of MCF-7 cells to doxorubicin (DOX) at doses inducing a strong G2 arrest resulted in a weak upregulation of p53. No site-specific phosphorylation of p53 at serine 46 was detected. These results indicate that p53 activation is dispensable for DOX-induced G2 arrest. Moreover, the pattern of p53 phosphorylation strongly depends on the type of the stimulating factor.

5-FdUrd-araC heterodinucleoside re-establishes sensitivity in 5-FdUrd- and AraC-resistant MCF-7 breast cancer cells overexpressing ErbB2.

ErbB2 overexpressing breast tumors have a poor prognosis and a high risk to develop chemoresistance to therapeutic treatment. "Chemoresistance" is a response of cells to toxic stress, and, although it is a common phenomenon, it is still poorly defined. However, a detailed understanding is required to target desensitized pathways and mechanisms for successful reactivation as part of a tailored therapy. To gain insight, which malfunctions contribute to chemoresistance, two mechanisms relevant for tissue homeostasis, the regulation of the cell cycle and of apoptosis, were investigated. Maternal MCF-7- and ErbB2-overexpressing MCF-7(erbB2) breast cancer cells were long term pretreated with 2'-deoxy-5-fluorodeoxyuridine (5-FdUrd) or 1-beta-d-arabinofuranosylcytosine (AraC) and the acquisition of drug-insensitivity was analyzed. A phosphate-conjugated heterodinucleoside consisting of one 5-FdUrd- and one AraC-moiety (5-fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine) was utilized as a tool to assess the type of acquired resistances. ErbB2-overexpression disrupted proper cell cycle regulation and furthermore facilitated the development of an apoptosis-refractory phenotype upon exposure to 5-FdUrd. Experiments with dimer 5-FdUrd-araC in ErbB2-overexpressing MCF-7(erbB2) cells, and also with nucleoside 5-FdUrd in maternal MCF-7 cells, evidenced that the phenotypes of resistance to cell cycle inhibition and to apoptosis induction were differently affected. The expression profile of cyclin D1 (but not that of p53, p21, or p27) correlated with the proliferative phenotypes and nuclear accumulation of apoptosis inducing factor (but not activation of caspase 7) with apoptotic phenotypes. Dimer 5-FdUrd-araC overrode acquired chemoresistances, whereas combined application of 5-FdUrd and AraC exhibited significantly less activity. Dimer 5-FdUrd-araC remained active in MCF-7 clones most likely by circumventing the prerequisite of first-step phosphorylation. The acquisition of chemoresistance encompassed the affection of apoptosis- and cell-cycle regulation to, respectively, different extents. Thus, drug-induced cell cycle arrest and apoptosis induction are independent of each other.

Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells.

We reported recently that roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrested human MCF-7 breast cancer cells in G2 phase of the cell cycle and concomitantly induced apoptosis. On the other hand, ROSC-induced G1 arrest observed by another group has not been accompanied by apoptosis. Therefore, we decided to prove to which extent components of tissue culture media could affect the primary action of ROSC. For this purpose we compared the efficacy of the ROSC treatment on MCF-7 cells cultivated in medium with and without phenol red. The kinetics of MCF-7 cell proliferation strongly depended on the presence of phenol red that has been recognized previously as a weak estrogen. Exposure of MCF-7 cells cultivated in phenol red-deprived medium to ROSC resulted in a strong G2 arrest and apoptosis. However, the anti-proliferative and pro-apoptotic action of ROSC was strongly diminished in cells maintained in medium containing phenol red. The ratio of the G2 cell population after 12 h ROSC was reduced by approximately 20% in the latter and correlated with the lack of CDK2 inactivation. Moreover, the kinetics of ROSC-induced apoptosis was delayed in the presence of phenol red. These results clearly evidence that the efficacy of the therapy of ER-positive breast cancers by CDK inhibitors is diminished in the presence of estrogen-mimicking compounds and indicate that phytoestrogens and xenoestrogens could interfere with the therapy. Therefore, the exposure of cancer patients to the estrogen mimics should be avoided at least during chemotherapy by CDK inhibitors.

Prevention of p53 degradation in human MCF-7 cells by proteasome inhibitors does not mimic the action of roscovitine.

We have recently observed activation of wild-type (wt) p53 protein in human MCF-7 breast cancer cells upon treatment with roscovitine (ROSC), a potent cyclin-dependent kinase inhibitor. It has been previously suggested that ROSC repressed transcription of Mdm-2, a negative p53 regulator, and that the lack of Mdm-2 contributes to the ROSC-induced upregulation of p53 protein. Therefore, we decided to see whether the prevention of p53 degradation by proteasome inhibitors will mimic the effects generated by ROSC. Exposure of human MCF-7 cells to different proteasome inhibitors resulted in a time-dependent increase of p53. However, unlike ROSC, they failed to modify p53 protein at Ser46 and to induce p53AIP1 protein. Moreover, whereas ROSC arrested MCF-7 cells in the G2-phase of the cell cycle, proteasome inhibitors blocked cells primarily in the S-phase, presumably because of the prevention of cyclin degradation. Our results indicate that prevention of p53 degradation by proteasome inhibitors does not mimic the action of ROSC.

A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death.

The efficacy of distinct anti-cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti-proliferative and pro-apoptotic action of a number of anti-cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti-proliferative and pro-apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose- and time-dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases-3/7 in two separate microtiter plates using the CellTiter-Glo Luminescent Cell Viability Assay and the Caspase-Glo 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter-Blue Cell Viability Assay and Caspase-Glo 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases-3/7. The new multiplexing assay offers a range of benefits over standard assays.

Roscovitine-induced up-regulation of p53AIP1 protein precedes the onset of apoptosis in human MCF-7 breast cancer cells.

We reported recently that roscovitine arrested human MCF-7 cancer cells at G2-M phase of the cell cycle and concomitantly induced apoptosis. After roscovitine treatment, the level of wild-type p53 protein strongly increased and p53 was accumulated in the nucleus. Here, we raised the question of which pathway would be involved in roscovitine-induced apoptosis in MCF-7 cells, which are known to be caspase-3-deficient, and whether roscovitine-mediated activation of p53 protein might positively affect the execution of cell death. Roscovitine induced a depolarization of mitochondrial potential beginning at 6 hours posttreatment as evidenced by changes in J-aggregate formation and release of the mitochondrial proteins cytochrome c and apoptosis-inducing factor. Interestingly, roscovitine stimulated a site-specific phosphorylation of wild-type p53 protein in a time-dependent manner. p53 protein was specifically phosphorylated at Ser46. P-Ser46-activated wild-type p53 tumor suppressor up-regulated p53AIP1 protein, its downstream target known to mediate the depolarization of mitochondria. The onset of phosphorylation of p53 at Ser46 preceded the up-regulation of p53AIP1 protein and the depolarization of mitochondrial potential. We compared the kinetics of roscovitine-mediated p53 activation between caspase-3-deficient parental MCF-7 cells and cells reconstituted with caspase-3. The kinetics and the extent of p53 protein activation in caspase-3-proficient cells differed from those observed in caspase-3-deficient parental cells. Remarkably, roscovitine failed to induce phosphorylation at Ser46 in caspase-3-reconstituted MCF-7 cells. Our results indicate that, depending on the status of caspase-3 in MCF-7 cells, different apoptotic pathways were initialized.

Increased susceptibility of poly(ADP-ribose) polymerase-1 knockout cells to antitumor triazoloacridone C-1305 is associated with permanent G2 cell cycle arrest.

Triazoloacridone C-1305 is a novel inhibitor of DNA topoisomerase II, which exhibits potent antitumor activity toward solid tumors. In this study, antiproliferative action of C-1305 and its close analog C-1533 was investigated in nontransformed mouse fibroblasts and two mutant cell lines in which the PARP-1 gene was specifically disrupted. Unexpectedly, C-1305 very strongly affected proliferation of cells lacking poly(ADP-ribose) polymerase-1 (PARP-1), whereas the action of less active compound C-1533 toward normal and PARP-1-negative cells was comparable. The IC(50) concentration of C-1305 determined for PARP-1 knockout cells was approximately 150-fold lower than that determined for cells with functional PARP-1. Both studied triazoloacridones exhibited very low direct cytotoxicity as evidenced by accumulation of 7-amino-actinomycin D, and only low levels of apoptosis were observed after a 24-h exposure to studied drugs. Analysis of DNA damage induced by C-1305 by the Comet assay showed that this drug induced very low levels of DNA strand breaks. C-1305 strongly affected cell cycle progression in normal and PARP-1 mutant cells and arrested both cell types in G(2)-M phase. However, the G(2)-M arrest induced by C-1305 was greatly prolonged in PARP-1-deficient cells as compared with normal fibroblasts. Together, these results show that mouse cells lacking PARP-1 are extremely sensitive to C-1305, a new topoisomerase II inhibitor. This is in striking contrast with previous reports in which PARP-1-deficient cells were shown to be resistant to classical topoisomerase II inhibitors. Our data also suggest that the PARP-1 status might be essential for the maintenance of the G(2) arrest induced by C-1305.

In vivo activated caspase-3 cleaves PARP-1 in rat liver after administration of the hepatocarcinogen N-nitrosomorpholine (NNM) generating the 85 kDa fragment.

We reported previously that treatment of rats with the hepatocarcinogen N-nitrosomorpholine (NNM) caused severe hepatotoxicity associated with apoptosis of hepatocytes beginning 12 h after administration of NNM. We observed that poly(ADP-ribose) polymerase 1 (PARP-1), one of the major nuclear targets for caspases, was proteolytically degraded generating primarily 64 and 54 kDa fragments. Interestingly, at 20, 30, and 40 h post-treatment a 85 kDa cleavage product of PARP-1 resembling that generated by caspase-3 appeared additionally in hepatocytes. More detailed analysis performed in the present study revealed that the 85 kDa fragment of PARP-1 was generated in the liver in 10 of 17 (60%) animals examined between 20 and 40 h after NNM administration. The caspase-3 generated 85 kDa fragment was detected solely in hepatocytes undergoing apoptosis as evidenced by immunostaining performed with the antibody recognizing exclusively PARP-1 cleaved at position 214/215. The appearance of the 85 kDa fragment of PARP-1 in the liver nuclei coincided temporally with an significant increase of caspase-3 activity in hepatocytes. In contrast, in testis samples obtained from the same animals, no changes characteristic for apoptosis such as induction of caspases activity or degradation of nuclear PARP-1 could be detected. Our results evidence unequivocally that PARP-1 in liver is not resistant to caspases and can be processed in vivo by activated caspase-3 producing the p85 kDa fragment. Moreover, the caspase-3 induced PARP-1 fragmentation coinciding with the increase of caspase-3 activity was detected solely in the target organ and exclusively in hepatocytes undergoing apoptosis. Considering the fact that the caspase-3 mediated PARP-1 cleavage occurred only in 60% of animals tested between 20 and 40 h, it becomes obvious that the cellular response in vivo to the same trigger(s) strongly varies and may depend on a variety of intrinsic factors. It remains to elucidate which additional factors may be involved in the modulation of cellular response to the strong insults thereby activating different pathways and generating distinct outcomes.

Non-apoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

We examined the action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on HeLa cells and compared it with that of cisplatin (CP). MNNG directly killed a substantial number of cells within 1 hour and resulted in strong DNA-damage as evidenced by Comet measurements. Despite appearance of DNA lesions, p53 protein was not activated. Analysis of HeLa cells treated with MNNG for 1h, 3h and 6h by flow cytometry and by Hoechst staining did not reveal any sub-G(1) cell population and chromatin condensation/fragmentation characteristic for apoptosis, respectively. Also, no biochemical changes typical for apoptosis such as activation of caspase-3 or release of cytochrome C from mitochondria were detected. Inactivation of PARP-1 reduced the direct cytotoxicity exerted by MNNG. Our results showing that despite appearance of severe DNA lesions after short exposure of HeLa cells to MNNG neither activation of p53 response nor induction of apoptosis occurred implicate that generation of strong DNA damage is not sufficient to stabilize p53 protein in HeLa cells. Our data unequivocally show that the conscientious determination of the type of cell death induced by genotoxic agents is necessary. The assessment of the changes based on at least a few independent criteria is required to discriminate between apoptosis and necrosis. Since the alkylating agents generate DNA strand breaks, the recruitment of methods based on determination of DNA cleavage such as DNA ladder or TUNEL assay for evaluation of apoptosis is not adequate.

Rapid onset of nucleolar disintegration preceding cell cycle arrest in roscovitine-induced apoptosis of human MCF-7 breast cancer cells.

The aim of our study was to explore the antiproliferative and pro-apoptotic action of roscovitine (ROSC) on human breast cancer MCF-7 cells. We examined the effect of ROSC on cell proliferation, cell cycle progression, nucleolar morphology, posttranslational modifications of histones as well as on induction of apoptosis. The effects of ROSC on the argyrophilic nucleolar organizer regions (AgNORs) and nucleolar RNA of MCF-7 cells were marked: ROSC treatment changed the pattern of AgNORs in a time-dependent manner. The disintegration of nucleoli manifested by increasing number of nucleolar fragments already began at 6 hr posttreatment. This was accompanied by a redistribution of the nucleolin from the nucleolus beginning after 6 hr and preceded a decrease of histone acetylation and phosphorylation. Inhibition of DNA synthesis and accumulation of G(2)/M-arrested cells starting 6 hr posttreatment coincided with a strong increase of the p53 level and with an appearance of a few cells committed to undergo apoptosis. However, all these changes preceded the main wave of apoptosis, which occurred after 24 hr ROSC treatment as assessed by determination of the frequency of Annexin binding, activation of caspases as well as of DNA fragmentation. Onset of PARP-1 cleavage detected by immunoblotting and by immunohistochemistry 6 hr or 9 hr posttreatment, respectively, preceded for a few hours the DNA fragmentation detected in situ by TUNEL assay. Reconstitution of MCF-7 cells with caspase-3 did not change the kinetics of ROSC-induced apoptosis. Our results show that disintegration of nucleoli is an early marker of ROSC-induced changes. Cell cycle arrest precedes the main wave of apoptosis.

Dual action of cyclin-dependent kinase inhibitors: induction of cell cycle arrest and apoptosis. A comparison of the effects exerted by roscovitine and cisplatin.

Cyclin-dependent kinases (CDKs) have recently raised considerable interest in view of their key role in the regulation of the cell cycle progression. In proliferating cells, distinct CDKs associated with specific cyclins coordinate in an orchestrated way the appropriate transition between different phases of the cell cycle. Mutations and/or aberrant expression of distinct CDKs and their regulatory components lead to uncontrolled proliferation and finally to carcinogenesis. However, in post-mitotic neurons, all CDKs with the exception of CDK5 are silent. CDK5, a proline-directed serine/threonine kinase exhibiting a close structural homology to the mitotic CDKs, binds to p35, the neuron-specific regulatory subunit of CDK5. CDK5 is very abundant in mature neurons and seems to regulate neurotransmitter release through phosphorylation and down-regulation of calcium channel activity. Therefore, the inhibition of CDKs in neurons after oxidative stress and in neurodegenerative disorders has a protective action. Selective CDKs inhibitors were developed as promising drugs for cancer therapy due to their ability to arrest cell cycle progression. The aim of this study was to compare the anti-proliferative effect of roscovitine (ROSC), a potent CDKs inhibitor, with that of cisplatin (CP) on human breast cancer MCF-7 cells. ROSC exerted stronger inhibitory effect on proliferation and cell cycle progression of MCF-7 than CP. Accumulation of G(2)/M arrested cells starting 6 h after onset of ROSC treatment coincided with a strong up-regulation of the p53. Reconstitution with caspase-3 sensitized MCF-7 cells to CP action. It implicates that ROSC inhibits more selectively and efficaciously the proliferation of human breast carcinoma cells.

MNNG induces dramatic DNA damage and non-apoptotic changes in cervical carcinoma HeLa cells.

It has been previously reported that a short treatment of human cervix carcinoma HeLa cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced apoptosis. We examined the action of MNNG on HeLa cells and compared it with that of cisplatin. MNNG damaged the integrity of the cell membrane and killed the cells within 3 hours. During this period no changes characteristic for apoptosis, such as cell shrinkage, condensation of nuclei, chromatin fragmentation or activation of caspases, could be detected. However, the exposure of HeLa cells to 50 micro M MNNG for 1 h resulted in dramatic DNA damage. The MNNG-induced disruption of cell membrane associated with cell death indicates that HeLa cells die by necrosis.