RESUMO
Expression of the PTEN tumor suppressor gene is abnormal in many human cancers. Loss of PTEN expression leads to the activation of downstream signaling pathways that have been associated with resistance to radiation. In non-small cell lung carcinoma (NSCLC), suppressed expression of PTEN is frequently due to methylation of its promoter region. In this study, we tested whether gene transfer of wild-type PTEN into an NSCLC cell line with a known methylated PTEN promoter, H1299, would increase its sensitivity to ionizing radiation. Pretreating H1299 cells with an adenoviral-mediated PTEN (Ad-PTEN)-expressing vector sensitized H1299 cells to radiation. To determine the mechanism responsible for radiosensitization, we first examined radiation-induced apoptosis, which was enhanced but did not correlate with radiosensitizing effect of Ad-PTEN. Therefore, we next examined the ability of Ad-PTEN to modulate the repair of radiation-induced DNA double-strand breaks (DSBs) using the detection of repair foci positive for gamma-H2AX, a protein that becomes evident at the sites of each DSB and that can be visualized by immunofluorescent staining. Compared with controls, the repair of radiation-induced DSBs was retarded in H1299 cells pretreated with Ad-PTEN, consistent with the radiosensitizing effect of the vector. We conclude that signal transduction pathways residing primarily in the cytoplasm may intersect with DNA damage and repair pathways in the nucleus to modulate cellular responses to radiation. Elucidating the mechanisms responsible for this intersection may lead to novel strategies for improving therapy for cancers with defective PTEN.
Assuntos
Adenoviridae/fisiologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Reparo do DNA/efeitos da radiação , Neoplasias Pulmonares/radioterapia , PTEN Fosfo-Hidrolase/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Linhagem Celular , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Neoplasias Pulmonares/terapia , PTEN Fosfo-Hidrolase/genética , Tolerância a Radiação , RadiossensibilizantesRESUMO
Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. p16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. HNSCC cells and xenografts (HPV/p16-positive and -negative) were used. p16-overexpressing and small hairpin RNA-knockdown cells were generated, and the effect of p16 on radiosensitivity was determined by clonogenic cell survival and tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction; and data from The Cancer Genome Atlas HNSCC project were analyzed. p16 overexpression led to downregulation of TRIP12, which in turn led to increased RNF168 levels, repressed DNA damage repair (DDR), increased 53BP1 foci and enhanced radioresponsiveness. Inhibition of TRIP12 expression further led to radiosensitization, and overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. These findings reveal that p16 participates in radiosensitization through influencing DDR and support the rationale of blocking TRIP12 to improve radiotherapy outcomes.
Assuntos
Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/virologia , Proteínas de Transporte/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/virologia , Papillomaviridae/fisiologia , Infecções por Papillomavirus/radioterapia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Biomarcadores Tumorais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Camundongos , Papillomaviridae/genética , Infecções por Papillomavirus/metabolismo , Tolerância a Radiação , Distribuição Aleatória , Carcinoma de Células Escamosas de Cabeça e Pescoço , Transfecção , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Current therapies used in the treatment of breast cancer are limited by systemic toxicity, rapid drug metabolism and intrinsic and acquired drug resistance. We have previously shown that adenoviral-mediated transfer of the melanoma differentiation-associated gene-7 (mda-7) elicits growth inhibition and apoptosis in various tumor types. Here, we evaluate the effects of Ad-mda7, alone and in combination with other therapies, against a panel of nine breast tumor cell lines and their normal counterparts; we report selective Ad-mda7-mediated p53-independent growth inhibition, G2/M cell cycle arrest, and apoptosis. In vivo, Ad-mda7 induced p53-independent tumor growth inhibition (P<0.004) in multiple xenograft models. We then evaluated the combination of Ad-mda7 with agents commonly used to treat breast cancer: radiotherapy (XRT), Tamoxifen, Taxotere, Adriamycin, and Herceptin. These agents exhibit diverse modes of action, including formation of bulky adducts, inhibition of DNA replication (Adriamycin, XRT), damage to microtubules (Taxotere), nonsteroidal estrogen antagonists (Tamoxifen), or Her2/neu receptor blockade (Herceptin). Treated with conventional anticancer drugs or radiation, MDA-7-expressing cells display additive or synergistic cytotoxicity and apoptosis that correlates with decreased BCL-2 expression and BAX upregulation. In vivo, animals that received Ad-mda7 and XRT underwent significant reduction of tumor growth (P<0.002). This is the first report of the synergistic effects of Ad-mda7 combined with chemotherapy or radiotherapy on human breast carcinoma cells.
Assuntos
Neoplasias da Mama/terapia , Carcinoma/terapia , Terapia Genética , Interleucinas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adenoviridae/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose , Terapia Biológica , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Carcinoma/tratamento farmacológico , Carcinoma/radioterapia , Terapia Combinada , Feminino , Técnicas de Transferência de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-bcl-2/análiseRESUMO
The cycle-dependent cytotoxicity of seven chemotherapy agents was compared with the use of subpopulations of Chinese hamster ovary cells separated into the various phases of the cell cycle by centrifugal elutriation. The proportion of cells killed by either beta-cytosine arabinoside or hydroxyurea agreed well with the proportion of S-phase cells in the treated subpopulations. Cell-cycle survival patterns were also determined for bleomycin (BLM), adriamycin (ADR), cis-diamminedichloroplatinum (II) (cis-DDP), Asaley, and Yoshi 864. Three of the agents (cis-DDP, Asaley, and Yoshi 864) had similar cell-cycle survival patterns in that they all preferentially killed cells in the G1 phase. In contrast, BLM preferentially killed cells in G2+M phase, whereas ADR was most cytotoxic to cells in middle to late S-phase. This investigation demonstrated that separation of cultured mammalian cells into the various cell-cycle phases by centrifugal elutriation allows the rapid determination of the phase-dependent cytotoxic effects exerted by chemotherapy agents.
Assuntos
Antineoplásicos/farmacologia , Ciclo Celular , Melfalan/análogos & derivados , Mesilatos/farmacologia , Animais , Bleomicina/farmacologia , Separação Celular/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisplatino/farmacologia , Cricetinae , Cricetulus , Citarabina/farmacologia , Doxorrubicina/farmacologia , Feminino , Hidroxiureia/farmacologia , Interfase/efeitos dos fármacos , Melfalan/farmacologia , Metáfase/efeitos dos fármacos , OvárioRESUMO
We have used the technique of alkaline elution to determine the amount of nitrogen mustard (HN2)-induced DNA cross-linking in a murine fibrosarcoma tumor in vivo. Mice were either treated with HN2 directly or were pretreated with misonidazole (MISO) or diethyl maleate prior to injection with HN2. Two types of HN2-induced DNA lesions were detected, namely, proteinase K-sensitive and -resistant cross-links. Pretreatment with MISO did not appear to affect the ratio of the two types of lesion. In mice treated with HN2 alone, the amount of cross-linking reached a high level by 0.5 hr postinjection, after which these lesions were repaired, 62% of cross-links being removed between 0.5 hr and 6 hr postinjection. Pretreatment of mice with MISO resulted in substantial alterations in both the magnitude and time course of cross-linking during the first few hr after injection of HN2. Both MISO and diethyl maleate enhanced the number of cross-links formed at 0.5 hr postinjection. Furthermore, in MISO-pretreated mice, only 18% of the cross-links present at 0.5 hr had been removed by 6 hr postinjection. This early enhancement is possibly related to glutathione depletion resulting in reduced intracellular inactivation of HN2. Since repair processes were determined not to be saturated at the level of lesions under study, these data suggest that, in addition to the initial glutathione depletion resulting in an increased burden of damage, MISO may also inhibit DNA repair processes, possibly via a hypoxia-dependent interaction between MISO reduction products and DNA or repair enzymes. Assuming that DNA cross-linking is related to the cytotoxicity of HN2, these effects may account for the MISO enhancement of HN2 toxicity toward various biological systems which have been reported previously. It appears that chemosensitization may result from a variety of factors, with the relative importance of each factor depending on the particular drug being used.
Assuntos
DNA de Neoplasias/metabolismo , Fibrossarcoma/fisiopatologia , Maleatos/farmacologia , Mecloretamina/toxicidade , Misonidazol/farmacologia , Nitroimidazóis/farmacologia , Animais , Esquema de Medicação , Cinética , Camundongos , Camundongos EndogâmicosRESUMO
Chinese hamster ovary cells were synchronized into the different phases of the cell cycle by centrifugal elutriation and treated with nitrogen mustard (HN2) in order to investigate the role of DNA damage and repair processes in the cell cycle-dependent cytotoxicity of this alkylating antitumor agent. In agreement with previous studies, cell populations enriched in G1 were the most sensitive to HN2, and those enriched in late S phase-G2 were more resistant, as determined by clonogenic assay. Although the variation in surviving fraction through the cell cycle in response to a single dose (3 micrograms/ml; 1.0 h) of HN2 was as great as a factor of 10, complete dose-response curves generated for the most sensitive and most resistant elutriator fractions indicated that such changes could be accounted for by a ratio of D0 values of only 1.4 Cells synchronized by this same method were also analyzed for their relative levels of HN2-induced DNA cross-linking using the sensitive technique of alkaline elution. There was no significant difference in the levels of either DNA interstrand or DNA-protein cross-links induced in the two elutriator fractions described above immediately after the HN2 treatment. When the amount of DNA cross-linking in the two fractions was measured 6 h after treatment, considerable repair had occurred; however, there was no measurable difference in the rate of repair of either type of cross-link (i.e., DNA interstrand and DNA-protein) in the different phases. Differences in DNA damage and repair processes could not therefore be resolved within the confidence limits of available assays and probably cannot account for the differential cytotoxicity of HN2 towards cells in the different cell cycle phases.
Assuntos
Alquilantes/toxicidade , Ciclo Celular/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/toxicidade , Animais , Linhagem Celular , Separação Celular/métodos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Reagentes de Ligações Cruzadas/toxicidade , Reparo do DNA , Feminino , Glutationa/metabolismo , OvárioRESUMO
The efficiency of DNA strand break formation in normal and tumor tissues of mice was measured using the technique of alkaline elution coupled with a microfluorometric determination of DNA. This methodology allowed measurement of the DNA strand breaks produced in tissues irradiated in vivo with doses of radiation comparable to those used in radiotherapy (i.e., 1.0 gray) without the necessity for the cells to be dividing and incorporating radioactive precursors to label the DNA. The results showed that substantial differences existed among various tissues in terms of the amount of DNA strand break damage produced for a given dose of radiation. Of the normal tissues, the most breaks were produced in bone marrow and the least were produced in gut. Furthermore, strand break production was relatively inefficient in the tumor compared to the normal tissues. The efficiency of DNA strand break formation measured in the cells from the tissues irradiated in vitro was much more uniform and considerably greater than that measured in vivo, suggesting that the normal tissues in the animal may be radiobiologically hypoxic.
Assuntos
Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos da radiação , DNA/efeitos da radiação , Fibrossarcoma/fisiopatologia , Animais , Medula Óssea/efeitos da radiação , Relação Dose-Resposta à Radiação , Intestino Delgado/efeitos dos fármacos , Cinética , Fígado/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos , Baço/efeitos da radiação , Distribuição TecidualRESUMO
To further understand the relationships between DNA damage, DNA repair, and cellular end points such as survival and mutation, the repair capacity of a DNA repair-deficient mutant (strain UV-20) of Chinese hamster ovary cells was characterized in response to DNA cross-linking agents. This mutant, previously shown to be hypersensitive to killing by both ultraviolet light and the cross-linking agent mitomycin C, was also found to be extremely sensitive to cis-diamminedichloroplatinum, another DNA cross-linking agent. The efficiency of DNA cross-link removal after treatment with mitomycin C or cis-diamminedichloroplatinum was measured using the technique of alkaline elution and compared in wild-type Chinese hamster ovary cells and strain UV-20. Wild-type cells removed 80 or 95% of the cross-links within 24 hr after treatment with cis-diamminedichloroplatinum or mitomycin C, respectively. In contrast, UV-20 cells, which were equally as susceptible to cross-link damage as were wild-type cells, removed only a small proportion of the cross-links made by either agent. These results emphasize the importance of DNA repair processes in modulating the cytotoxic effects of chemicals that produce DNA cross-link damage and suggest that cross-link repair in Chinese hamster ovary cells is controlled by a pathway that also repairs damage from ultraviolet radiation.
Assuntos
Reparo do DNA , DNA/genética , Mutação , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fenômenos Químicos , Química , Cisplatino/farmacologia , Cricetinae , Cricetulus , Feminino , Cinética , Mitomicina , Mitomicinas/farmacologia , OvárioRESUMO
The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Zinostatina/toxicidade , Animais , Radioisótopos de Carbono , Linhagem Celular , Cricetinae , Cricetulus , DNA/genética , DNA/efeitos da radiação , Feminino , Cinética , Mutação , OvárioRESUMO
The formation and repair of cis-diamminedichloroplatinum(II) (cis-DDP)-induced DNA cross-links in cells from a number of different mouse tissues, both normal and neoplastic, were compared in three different populations of animals, tumor-free mice and mice bearing a transplanted fibrosarcoma (either FSa or NFSa) in their thighs. Groups of mice were given i.v. injections of 4-12-mg/kg doses of cis-DDP, and the amount of cis-DDP-induced DNA cross-linking was determined at different times after injection using an adaptation of the alkaline elution technique. The degree of cross-linking in each tissue was linearly related to the dose of cis-DDP at either 6 or 24 h after injection and varied significantly among the different tissues, with FSa, NFSa, kidney, and liver showing the highest level of cross-linking of the tissues studied. The relative contributions of DNA-interstrand and DNA-protein cross-links to the elution profiles were estimated by proteinase K (PK) digestion. At either 6 or 24 h after injection with cis-DDP, the rate of elution of the DNA was substantially increased by PK, indicating a large contribution of DNA-protein cross-links. This effect was observed in all tissues studied, although the proportion of PK-resistant lesions appeared to vary from tissue to tissue, liver and spleen showing a significantly lower proportion of DNA-interstrand to total cross-links than either of the tumors. For liver, virtually no interstrand cross-links could be detected after PK treatment. The kinetics of the repair of cis-DDP-induced DNA cross-linking in these tissues were also compared. In cells from tumor-free animals, the amount of total (DNA-interstrand plus DNA-protein) cross-linking increased gradually, reaching a maximum after about 6 h; however, little evidence of repair of these lesions was observed in any of these normal tissues. In fact, the degree of cross-linking tended to increase somewhat between 6 and 24 h after injection. The kinetics of cross-linking in cells isolated from the FSa tumor were very different; while there was an initial increase in cross-linking up to 6 h, these lesions were subsequently repaired, although at a somewhat slower rate than has been reported for cultured mammalian cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cisplatino , Reagentes de Ligações Cruzadas , DNA , Animais , Células Cultivadas , Cricetinae , Reparo do DNA , DNA de Neoplasias , Cinética , CamundongosRESUMO
The mechanism of action of 6-thioguanine (TG) has been examined in cultured Chinese hamster ovary cells by direct measurement of the incorporation of the compound into DNA and by analysis of the resulting DNA damage. The predominant lesions as monitored by alkaline elution were DNA strand breaks. Very few, if any, interstrand or DNA-protein cross-links could be definitively observed. The cytotoxicity of TG as measured by colony-forming ability appeared closely related with its incorporation into DNA and the DNA strand scission events. As TG concentrations were increased, cytotoxicity, DNA incorporation, and strand scission reached a plateau; this result is consistent with earlier reports that TG produces a reversible block of DNA synthesis. Strand breaks appeared to be related to the incorporation of TG into DNA, since the addition of 1 microM cycloheximide during a 24-hr treatment with 3 microM TG prevented the cytotoxicity, prevented incorporation of TG into DNA, and eliminated the strand breaks. Alkali-labile sites were detected in the DNA of TG-treated cells by alkaline elution at pH 12.8, suggesting that depurination of TG residues by a glycosylase mechanism may occur. It is also postulated that TG residues are recognizable by the long-patch repair system, since UV-sensitive cells deficient for long-patch repair were more sensitive to TG than were wildtype cells. Furthermore, caffeine (1 mM) was shown to enhance the lethality of TG (3 microM), as monitored by colony formation, without altering levels of TG incorporation into DNA or the strand scission as measured immediately after treatment. This result, coupled with the known delayed cytotoxic response of TG, suggests that gaps may occur in newly synthesized DNA opposite TG residues and that the repair of these gaps by a postreplication repair mechanism is inhibited by caffeine.
Assuntos
Replicação do DNA/efeitos dos fármacos , DNA/metabolismo , Tioguanina/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Feminino , Cinética , Ovário , RNA/biossínteseRESUMO
The cytotoxic effects of cis-diamminedichloroplatinum(II) (cis-DDP) were shown to be strongly potentiated by hyperthermia. The molecular mechanisms responsible for this potentiation were investigated by assaying the degree of DNA cross-linking produced under the different drug treatment conditions by the technique of alkaline elution. The results showed that the cells treated with the drug at 43 degrees had a greater amount of DNA cross-linking immediately after treatment than did cells treated with the drug at 37 degrees, indicating a possible thermal enhancement of drug uptake by the cells. Whereas the hyperthermia potentiated the cytotoxicity of cis-DDP by a factor of nearly 10, the degree of DNA cross-linking was only enhanced by a factor of 6.5, suggesting that while a large portion of the enhanced cytotoxicity may be attributed to the increased cross-linking other factors may also play some role. The possible influence of hyperthermia on the repair of the DNA damage induced by cis-DDP was investigated; however, no significant differnence in the rate of disappearance of cross-links between cells treated at 37 or 43 degrees was observed. Advantageous combination of chemotherapy with cis-DDP and hyperthemia for the treatment of cancer is implied by these results.
Assuntos
Cisplatino/farmacologia , DNA/metabolismo , Temperatura Alta , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Reagentes de Ligações Cruzadas , Reparo do DNA/efeitos dos fármacos , Feminino , Temperatura Alta/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/terapia , OvárioRESUMO
Centrifugal elutriation was used to separate cells dissociated from two hypotetraploid mouse solid tumors, a fibrosarcoma and a sarcoma derived from L-P59 cells, based on their sedimentation rates. The separation was rapid, requiring less than 1 hr; yielded about 80 percent cell recovery; and resulted in little loss of cell viability. Aanlysis of DNA content by flow cytometry demonstrated the synchrony obtained with these tumor cells. The fractions with the lowest sedimentation rates contained predominantly normal cells, those with intermediate sedimentation rates contained predominantly tumor cells in the G1 phase of the cell cycle, and those with the highest sedimentation rates contained mostly tumor cells in S or G2. The clonogenicity of L-P59 cells, assayed in culture, markedly increased with increasing sedimentation rates. In contrast, the clonogencity of fibrosarcoma cells, assayed in vivo by a lung colony assay, was lower for the smaller cells, but was essentially constant among the larger cells. Autoradiography of cells labeled in vivo with tritiated thymidine demonstrated no differences in the proportions of cycling cells in various fractions. These results demonstrate that subpopulations differing in cell type, phase of the cell cycle, and clonogencity can be rapidly separated from solid tumors by centrifugal elutriation.
Assuntos
Separação Celular/métodos , Sarcoma Experimental , Animais , Contagem de Células , Ciclo Celular , Sobrevivência Celular , Centrifugação , Células Clonais/patologia , Feminino , Fibrossarcoma/patologia , Camundongos , Camundongos Endogâmicos C3H , Sarcoma Experimental/patologiaRESUMO
We examined the influence of adenovirus-mediated wild-type p16INK4a (Ad/p16) expression on the radiation sensitivity of NSCLC cell lines, all of which lacked constitutive p16INK4a but each of which varied in p53 status: A549 (-p16INK4a/ +pRb/wt-p53), H322 (-p16INK4a/ +pRb/mt-p53), and H1299 (-p16INK4a/ +pRb/deleted-p53). The in vitro clonogenic survival results indicate that Ad/p16 enhanced the radiosensitivity of A549 but not H322 or H1299. Further analysis indicated that the apoptosis induced by combination therapy using Ad/p16 plus irradiation was dependent on the endogenous p53 status of the cancer cells. We performed Western blotting to analyse the p53 protein expression of A549 cells treated with either Ad/p16 or Ad/Luc. Endogenous p53 protein levels were higher in A549 cells transfected with Ad/p16 than in those transfected with Ad/Luc. Furthermore, when wt-p53 protein expression was restored in H1299 using Ad/ p53, Ad/p16 stabilized p53 protein expression and radiosensitized the cells. These results suggest that Ad/ p16-induced stabilization of p53 protein may play an important role in Ad/p16 mediated radiosensitization by enhancing or restoring apoptosis properties. Thus, Ad/ p16 plus radiation in combination may be a useful gene therapy strategy for tumors that have wt-p53 but nonfunctional p16INK4a.
Assuntos
Apoptose/efeitos da radiação , Proteínas de Transporte/fisiologia , Tolerância a Radiação/efeitos da radiação , Proteína Supressora de Tumor p53/biossíntese , Adenovírus Humanos , Western Blotting/métodos , Carcinoma Pulmonar de Células não Pequenas , Proteínas de Transporte/genética , Inibidor p16 de Quinase Dependente de Ciclina , Expressão Gênica , Vetores Genéticos , Humanos , Neoplasias Pulmonares , RNA Mensageiro , Ribonucleases/metabolismo , Células Tumorais CultivadasRESUMO
To determine if TRAIL-induced apoptosis in human prostate tumor cells was suppressed by bcl-2, we compared the levels of apoptosis induced by recombinant human TRAIL in pairs of isogenic cell lines that do or do not express bcl-2. Three human prostate tumor cell lines (PC3, DU145 and LNCaP) and their bcl-2-expressing counterparts were tested for their susceptibility to TRAIL. Cells were exposed to TRAIL in the presence of cycloheximide which acted as a sensitizer. Apoptosis was induced rapidly in PC3 and DU145 neo-control transfected cells, whereas induction in LNCaP required 24 h. All three cell line variants expressing bcl-2 were resistant to the apoptotic effects of TRAIL. Caspase 3 and 8 activation was also detected in the neo control cells after treatment with TRAIL, demonstrating the rapid activation of the caspase cascade similar to that seen with other death receptors. Bcl-2 overexpression in these cells blocked activation of these caspases, suggesting that bcl-2 expression of human cancer cells may be a critical factor in the therapeutic efficacy of TRAIL.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Antineoplásicos/metabolismo , Proteínas Reguladoras de Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Cicloeximida/farmacologia , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Humanos , Ligantes , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The mechanism by which Bcl-2 oncogene expression inhibits radiation-induced apoptosis has been investigated in two mouse lymphoma cell lines: line LY-as is radiation sensitive, displays substantial radiaton-induced apoptosis, and expresses low levels of Bcl-2; line LY-ar is radiation-resistant, displays a low apoptosis propensity, and expresses 30-fold higher amount of Bcl-2 protein than does the sensitive line. We observed that upon incubation in cystine/methionine-free (C/M-) medium, radiation-induced apoptosis in the LY-ar cells was restored to levels comparable to that seen in the LY-as cells. lntracellular glutathione (GSH) concentrations in LY-ar cells incubated in C/M- medium plummeted to 50% of control values within 2 h. LY-ar cells treated with diethyl maleate (DEM) or diamide, agents that deplete cellular thiols, had increased susceptibility to radiation-induced apoptosis in a manner similar to C/M- medium. These results are consistent with the general idea that Bcl-2 expression blocks apoptosis through an antioxidant pathway that involves cellular thiols. That Bcl-2-expressing tumor cells can be sensitized by exogeneous agents that modify cellular thiols offers strategies for overcoming such resistance.
Assuntos
Apoptose/fisiologia , Genes bcl-2 , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Tolerância a Radiação , Animais , Apoptose/efeitos da radiação , Butionina Sulfoximina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Fragmentação do DNA , Relação Dose-Resposta à Radiação , Glutationa/metabolismo , Cinética , Linfoma , Camundongos , Proteínas Proto-Oncogênicas/biossíntese , Fatores de Tempo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossíntese , Proteína X Associada a bcl-2RESUMO
Multistep lymphomagenesis involves the deregulation of oncogenes and inactivation of tumor suppressor genes resulting in altered rates of proliferation as well as apoptotic cell death in tumor cells. The contribution of bcl-2 and p53 to the regulation of cell death during lymphomagenesis is assessed using bcl-2-1g, p53 'knock-out' (p53 KO), and p53 KO/bcl-2 hybrid mice. PCR-SSCP and DNA sequence analysis demonstrated that p53 somatic mutations are uncommon in lymphomas arising in bcl-2-Ig transgenic mice. Reduction in tumor latency was not observed in p53 KO/bcl-2 hybrid mice compared to p53 KO mice. Furthermore, overexpressed bcl-2 suppressed wild-type p53 associated apoptosis following gamma-radiation. These findings indicate that bcl-2 and p53 serve a suppressor and effector function, respectively, of a common cell death pathway. These findings also suggest that p53 somatic mutations provide no selective advantage during in vivo multistep lymphomagenesis in the context of bcl-2 gene deregulation.
Assuntos
Apoptose/genética , Linfoma/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Sequência de Bases , Dano ao DNA/genética , Primers do DNA , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
The influence of the nuclear ADP-ribosyltransferase inhibitor 3-aminobenzamide on the DNA strand-break rejoining kinetics and cytotoxicity in Chinese hamster ovary cells following H2O2 treatment was investigated. For the DNA damage studies, cells were treated on ice with H2O2 (0-20 microM) for 1 h in serum-free medium, after which the H2O2 was removed and the cells were allowed to repair their damage in complete medium at 37 degrees C in the presence or absence of 3-aminobenzamide (5 mM) for periods up to 2 h. The DNA strand breaks remaining as a function of time were then estimated by alkaline elution. A linear relationship between the H2O2 concentration and the initial level of DNA single-strand breaks (zero time allowed for repair) was observed. No double-strand breaks or DNA-protein cross-links were detected at these doses. The rejoining of single-strand breaks after H2O2 (20 microM) alone was characterized by a single exponential process with a t1/2 of approx. 5 min. However, in the presence of 3-aminobenzamide, rejoining was much slower and biphasic, with t1/2 of approx. 10 and 36 min. The inhibitory action of 3-aminobenzamide was concentration-dependent and completely reversible in that, when the 3-aminobenzamide was removed from the treated cultures, the strand-break rejoining kinetics rapidly returned to the t1/2 of 5 min typical of H2O2 alone. Considerably higher concentrations of H2O2 (up to 600 microM) were required for cell killing compared to the DNA damage studies. Cell killing by H2O2 alone was characterized by a shoulderless, exponential survival curve (D0 = 880 microM). The cytotoxicity was potentiated when the cells were treated with 3-aminobenzamide (5 mM) for 1 h after the H2O2 treatment; the survival curve with 3-aminobenzamide also assumed a biphasic character (D0 of 212 microM and 520 microM). These results are consistent with the theory that OH.-induced single-strand breaks do not normally represent lethal lesions to the cell because of their rapid, efficient repair. However, interference with these repair processes (in this case by 3-aminobenzamide) can alter this relationship, possibly allowing lesion fixation.
Assuntos
Benzamidas/farmacologia , DNA/metabolismo , Animais , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Fibroblastos/análise , Fibroblastos/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , OvárioRESUMO
We examined the effect of exposure to H2O2 at 37 degrees C on Chinese hamster ovary cell survival, DNA single-strand break (SSB) induction and rejoining, and activation of poly(ADP-ribose) (ADPR) polymerase. The effect of the ADPR polymerase inhibitor 3-aminobenzamide on each of these processes was also determined. SSB induction increased progressively with increasing H2O2 concentration. SSB levels were maximal after approx. 5 min of exposure to H2O2 (100 microM) and then decreased at longer times. This decrease, which paralleled the time-dependent depletion of H2O2, was due to the rejoining of SSBs. 3-Aminobenzamide enhanced the level of SSBs at each time point. H2O2 increased the level of both ADPR synthesis and NAD+ depletion (both measures of ADPR polymerase activity) in a concentration-dependent fashion, with the maximum effect being reached after approx. 20 min. After 100 microM H2O2, the effects on both ADPR and NAD+ were reversible. 3-Aminobenzamide completely blocked the effects of the oxidant on both NAD+ and ADPR levels. Thus, SSB induction by H2O2 at 37 degrees C was accompanied by a marked but reversible stimulation of ADPR polymerase. However, cell killing by H2O2 was only slightly enhanced in the presence of 3-aminobenzamide (5 mM), so the above-mentioned effects do not appear to be relevant to the cytotoxic effect of H2O2 under these conditions. Comparing these results with data obtained previously for cells treated with H2O2 at 4 degrees C suggests that the mechanisms of DNA strand breakage and cell killing may be quite different at the two temperatures, and that DNA damage at 37 degrees C may be indirectly mediated by temperature-dependent metabolic events.
Assuntos
Dano ao DNA , DNA/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Açúcares de Nucleosídeo Difosfato/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , NAD/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de TempoRESUMO
In this study, we investigated the importance of redox and Bcl-2 status on cytochrome c-mediated apoptosis. Two mouse lymphoma cell lines, LYas and LYar that express Bcl-2 protein at different levels, were used to reconstitute a cell-free system. Cytoplasmic extracts made from apoptosis-sensitive LYas cells 2.5 h after exposure to 5 Gy gamma-radiation were able to induce apoptosis in isolated nuclei, whereas extracts made from LYas cells at time points earlier than 2. 5 h, or from Bcl-2-overexpressing, apoptosis-resistant LYar cells at all time points after irradiation were inactive. Apoptotic activity was restored to inactive extracts by the addition of oxidized but not reduced cytochrome c. Cytochrome c reductase was able to inhibit apoptosis in extracts made from LYas cells 2.5 h after irradiation and LYar extracts activated by addition of oxidized cytochrome c. Antioxidants, but not oxidant defensive enzymes, blocked apoptosis implying that antioxidants might alter the redox state of factors important in mediating apoptosis. These findings confirm the importance of cellular redox state during apoptosis and are consistent with a role for Bcl-2 in regulating this redox state.