RESUMO
BACKGROUND: Hypoxia is thought to be an adverse feature of pancreatic cancer, but direct measurement in patients is technically challenging. To address this, we characterised the intra/interpatient heterogeneity of hypoxia in surgical specimens from patients who received the 2-nitroimidazole tracer pimonidazole pre-operatively. METHODS: Pimondazole was given intravenously 16-20 h before pancreatectomy, and the extent and intratumoral heterogeneity of hypoxia determined by image analysis applied to multiple tissue blocks stained by immunohistochemistry. Intra/interpatient heterogeneity was estimated by variance component analysis. RESULTS: Pimonidazole staining was analysed in 10 tumours. The extent of labelling varied amongst patients (0-26%), with a broader range of hypoxia in the epithelial (1-39%) compared with the stromal (1-13%) compartments. Variance component analysis demonstrated greater inter- than intrapatient variability of hypoxia, and that multiple (4-5) tumour sections are required to provide a consistent evaluation of its extent in individual tumours. CONCLUSIONS: There is significant intra- and intertumoral heterogeneity of hypoxia in pancreatic cancers, and these do not appear to be generally more hypoxic than other cancer types. This study establishes the feasibility to assess hypoxia in pancreatic cancer patients using pimonidazole, but questions the reliability of measurements made using a single tissue section.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Hipóxia Celular , Indicadores e Reagentes/metabolismo , Nitroimidazóis/metabolismo , Neoplasias Pancreáticas/metabolismo , Adulto , Análise de Variância , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Estudos de Viabilidade , Feminino , Humanos , Imuno-Histoquímica , Indicadores e Reagentes/administração & dosagem , Injeções Intravenosas , Masculino , Nitroimidazóis/administração & dosagem , Pâncreas/metabolismo , Pancreatectomia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Pré-Medicação , Viés de SeleçãoRESUMO
BACKGROUND: Germline mutations of the BRCA tumour suppressors have been associated with increased risk of pancreatic cancer. Clinical evidence suggests that these patients may be more sensitive to treatment with cisplatin. As the frequency of germline BRCA mutations is low, definitive experimental data to support the clinical observations are still missing. METHODS: We tested gemcitabine and cisplatin sensitivity of four BRCA1 and BRCA2 mutant and three BRCA1 and BRCA2 wild-type (WT) patient-derived pancreatic cancer xenografts. RESULTS: We observed treatment sensitivity to gemcitabine and cisplatin in the BRCA WT and mutant models. The BRCA1 and BRCA2 mutant xenografts were significantly more sensitive to cisplatin although these models also showed sensitivity to gemcitabine. The BRCA1 and BRCA2 WT models showed sensitivity to gemcitabine but not cisplatin. Treatment sensitivity in the xenograft models closely resembled treatment response in the corresponding patients. DISCUSSION: We have characterised a panel of xenografts derived from pancreatic cancer patients carrying germline BRCA mutations, and shown that their genetic features resemble the patient donor. Our results support further clinical testing of treatment regimens combining gemcitabine and platinum drugs in this patient population, as well as preclinical research aiming to identify mechanisms of cisplatin resistance in BRCA mutant pancreatic cancers.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Genes BRCA1 , Genes BRCA2 , Mutação em Linhagem Germinativa , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
The phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway is frequently deregulated in pancreatic cancers, and is believed to be an important determinant of their biological aggression and drug resistance. NVP-BEZ235 is a novel, dual class I PI3K/mammalian target of rapamycin (mTor) inhibitor undergoing phase I human clinical trials. To simulate clinical testing, the effects of NVP-BEZ235 were studied in five early passage primary pancreatic cancer xenografts, grown orthotopically. These tumours showed activated PKB/Akt, and increased levels of at least one of the receptor tyrosine kinases that are commonly activated in pancreatic cancers. Pharmacodynamic effects were measured following acute single doses, and anticancer effects were determined in separate groups following chronic drug exposure. Acute oral dosing with NVP-BEZ235 strongly suppressed the phosphorylation of PKB/Akt, followed by recovery over 24 h. There was also inhibition of Ser235/236 S6 ribosomal protein and Thr37/46 4E-BP1, consistent with the effects of NVP-BEZ235 as a dual PI3K/mTor inhibitor. Chronic dosing with 45 mg kg(-1) of NVP-BEZ235 was well tolerated, and produced significant tumour growth inhibition in three models. These results predict that agents targeting the PI3K/Akt/mTor pathway might have anticancer activity in pancreatic cancer patients, and support the testing of combination studies involving chemotherapy or other molecular targeted agents.
Assuntos
Imidazóis/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Quinolinas/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Antineoplásicos/uso terapêutico , Diferenciação Celular , Criança , Inibidores Enzimáticos/uso terapêutico , Humanos , Imuno-Histoquímica , Camundongos , Camundongos SCID , Neoplasias Pancreáticas/patologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Serina-Treonina Quinases TOR , Transplante HeterólogoRESUMO
Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using in vitro kinase assays, we found that EphA2 receptor tyrosine kinase was inhibited directly by dasatinib in a dose-dependent manner. Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src.
Assuntos
Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptor EphA2/antagonistas & inibidores , Transdução de Sinais , Tiazóis/farmacologia , Animais , Linhagem Celular Tumoral , Dasatinibe , Humanos , Masculino , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Neoplasias Pancreáticas/enzimologia , Receptor EphA2/metabolismoRESUMO
The cytotoxicity of idoxuridine (IdUrd), a thymidine analogue, and ICI D1694 (D1694), a folate-based thymidylate synthase (TS) inhibitor, were examined individually and in combination in two human tumor cell lines. MGH-U1 bladder cancer and HCT-8 colon cancer cells were grown as monolayer cultures with and without thymidine. The cytotoxicity of these agents alone and in combination were determined using normal human bone marrow colony-forming unit, granulocyte-macrophage (CFU-GM) as a surrogate for myelosuppression in vivo. Thymidylate synthase inhibition, IdUrd incorporation into DNA, and DNA single-strand breaks were measured in each cell line and related to cytotoxicity. The cytotoxicity of a 24-h exposure to IdUrd or D1694 increased with drug concentration in each cell line. The drug concentrations producing 50% and 10% clonogenic survival in MGH-U1 cells, respectively, were 0.006 and 0.009 microM for D1694 and 13.0 and 81.0 microM for IdUrd. Those for HCT-8 cells, respectively, were 0.009 and 0.018 microM for D1694 and 7.5 and 20.5 microM for IdUrd. The cytotoxicity of IdUrd combined with D1694 was synergistic in both MGH-U1 and HCT-8 cells as determined by median-effect analysis. The addition of thymidine at concentrations of 0.1, 0.3, and 1.0 microM to the culture medium did not decrease the cytotoxicity of D1694 in either tumor cell line. TS inhibition using the whole cell assay was observed with only D1694, producing 50% inhibition of TS activity at 0.002 microM for MGH-U1 and 0.007 microM for HCT-8 cells. IdUrd did not inhibit TS activity, nor did it enhance the TS inhibitory effects of D1694. The incorporation of IdUrd into DNA increased with increasing concentrations of D1694. This increased DNA incorporation correlated with the increase in DNA single-strand breaks. DNA single-strand breaks paralleled cytotoxicity. CFU-GM survival, exposed to the same drug concentrations as those used in the tumor cell lines, revealed that the therapeutic index was greater for the combination than for either agent alone. These findings suggest that IdUrd plus D1694 is a promising new drug combination, which may have a favorable therapeutic index in vivo.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Idoxuridina/farmacologia , Quinazolinas/farmacologia , Tiofenos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Sinergismo Farmacológico , Humanos , Idoxuridina/metabolismo , Células Tumorais CultivadasRESUMO
DNA index and S-phase fraction of 490 primary breast carcinomas were measured by flow cytometry, using paraffin-embedded tissue as a starting point. All patients had involvement of axillary lymph nodes and all were randomized onto one of the Ludwig Breast Cancer Trials I-IV between July 1978 and August 1981. The presence of an abnormal DNA stemline correlated with involvement of four or more axillary lymph nodes (P = 0.09), postmenopausal status (P = 0.008), estrogen receptor negativity (P = 0.03), and high tumor grade (P = 0.0005). Disease-free and overall survival for these patients was worse than for those with tumors of normal DNA content, but DNA aneuploidy did not have independent prognostic significance when allowance was made for its correlation with other prognostic features. The percentage of cells in S phase, which is an index of cell proliferation, was also analyzed in 285 DNA histograms considered to be capable of giving a valid estimate. Disease-free survival for 154 patients with S phase less than or equal to 10% was significantly longer than for those with S phase greater than 10% (P = 0.0008). S-phase greater than 10% was strongly correlated with high tumor grade (P less than 0.00001) and abnormal DNA index (P less than 0.00001) but only weakly correlated with nodal, hormone receptor, and menopausal status. Multivariate analysis using a Cox proportional hazard model suggested that the prognostic significance of the percentage of S phase was related to the association with tumor grade. Because it gives rapid, objectively determined information about biological aggressiveness DNA flow cytometry may well establish a role in routine clinical practice, but further technical refinements are required before it can be used for treatment decision making in nodes positive early breast cancer patients.
Assuntos
Neoplasias da Mama/análise , DNA de Neoplasias/análise , Interfase , Feminino , Citometria de Fluxo , Humanos , Metástase Linfática , PloidiasRESUMO
Human pancreatic adenocarcinoma cell lines PK1 and PK8 are resistant to the clinically relevant chemotherapy agent gemcitabine. Cell cycle analysis demonstrated an accumulation of cells in the early S phase during treatment with 20 microM gemcitabine, consistent with its mode of action as a DNA chain terminator. However, apoptosis was evident in only a small percentage of cells. Similar to pancreatic cancers in the clinic, PK1 and PK8 cells carry constitutively active Ki-Ras and overexpress multiple receptor tyrosine kinases. Both genetic abnormalities may potentially up-regulate the activity of the phosphatidylinositide 3-kinase (P13K)-protein kinase B (PKB)/Akt cell survival pathway. The current study examined the relevance of this pathway in the modulation of drug resistance in PK1 and PK8 cells. After exposure to 20 microM gemcitabine for 48 h and in the continuous presence of the drug, treatment with the P13K inhibitors wortmannin (50-200 nM) and LY294002 (15-120 microM) for 4 h substantially enhanced apoptosis in a concentration-dependent manner as compared with treatment with gemcitabine alone, as determined by the loss of mitochondrial membrane potential and the increase in propidium iodide uptake using flow cytometry. Furthermore, Western blotting showed that the reduction of phosphorylated PKB/Akt levels correlated with the enhancement of gemcitabine-induced apoptosis, suggesting that the PI3K-PKB/Akt pathway plays a significant role in mediating drug resistance in human pancreatic cancer cells. PI3K inhibitors may have therapeutic potential when combined with gemcitabine in the treatment of pancreatic cancers.
Assuntos
Adenocarcinoma/enzimologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias Pancreáticas/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Androstadienos/farmacologia , Cromonas/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Morfolinas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Wortmanina , GencitabinaRESUMO
The effects of de novo dTMP inhibition by N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2- thenoyl)-L-glutamic acid (D1694) or N6-[4-(morpholinosulfonyl)benz]-N6-diaminobenz[cd]indole glucuronate (AG-331) on clonogenic survival, thymidylate synthase (TS) and thymidine kinase (TK) activity, and expression of S-(p-nitrobenzyl)-6-thioinosine-sensitive nucleoside transporter (NT) sites were addressed in the human bladder cancer cell line, MGH-U1. These two TS inhibitors are structurally diverse. D1694 is a folate-based TS inhibitor, whereas AG-331 is a novel agent that inhibits the cofactor binding site of the enzyme. They also differ with respect to their cytotoxic effects in this cell line; D1694 cytotoxic 50% inhibitory concentration (IC50) and IC90 were 6.0 and 9.0 nM, respectively and IC50 and IC90 for TS inhibition were 2.5 and 4.8 nM, respectively. In contrast, AG-331 cytotoxic IC50 could not be achieved even at concentrations of up to 20 microM for 24-h exposures, and IC50 and IC90 for TS inhibition were 0.7 and 3.0 microM, respectively. Similar effects for D1694 and AG-331 were observed in their modulation of TK activity and NT expression. 5-(SAENTA-x8)-Fluorescein, a highly modified form of adenosine incorporating a fluorescein molecule which binds with a 1:1 stoichiometry to S-(p-nitrobenzyl)-6-thioinosine-sensitive NT sites, was used to investigate the expression of NT following exposure of cells to D1694 and AG-331. TK activity was addressed by the metabolism of [3H]thymidine to [3H]TMP by cellular extracted protein and by an alternative flow cytometric method using a modified form of thymidine incorporating a fluorescent molecule, dansyl-5-amino-2-deoxyuridine. Results obtained by both methods were comparable. At concentrations of 5 and 10 nM, D1694 increased TK activity 2.3-4.5-fold and NT expression 34-39-fold. AG-331, at concentrations of 5 and 10 microM, increased TK activity 1.8-2.5-fold and NT expression 22-31-fold, respectively. These data suggest that TK activity and NT expression have a common regulatory mechanism which is sensitive to endogenous dTTP pools and that the salvage pathway is a complex system of kinases coordinated with transport of nucleosides.
Assuntos
Proteínas de Transporte/metabolismo , Indóis/farmacologia , Proteínas de Membrana/metabolismo , Quinazolinas/farmacologia , Tiofenos/farmacologia , Timidina Quinase/biossíntese , Timidilato Sintase/antagonistas & inibidores , Humanos , Proteínas de Transporte de Nucleosídeos , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/metabolismoRESUMO
We have studied the antiproliferative effects of gallium nitrate in cultured CCRF-CEM lymphoblasts. The 50% inhibitory dose for these cells was 120 microM, and after 24 h at a cytostatic concentration (480 microM) S-phase arrest was observed by DNA flow cytometry. Deoxyribonucleoside triphosphate pools were all reduced (dATP, dGTP, and dCTP by 50%, dTTP by 25%), suggesting inhibition of ribonucleotide reductase. Administration of tracer amounts (0.5 microM) of either [3H]uridine or [3H]deoxyuridine confirmed that DNA synthesis had been inhibited to 20% of control rates by gallium. Further, the flow of the ribonucleoside into the dTTP pool and DNA was selectively reduced compared to that of the deoxyribonucleoside. Gallium decreased the specific activity of dTTP labeled from uridine by 50%, whereas the specific activity of dTTP labeled from deoxyuridine was increased 2.5-fold. Thus counts in DNA derived from [3H]uridine were decreased by more than 80%, while counts in DNA derived from [3H]deoxyuridine were virtually unaltered. Uridine incorporation into RNA was not affected. Gallium did not significantly alter the capacity of permeabilized naive cells to incorporate [3H]dTTP into DNA, while 24-h gallium pretreatment (which increased the percentage of S-phase cells) produced a modest increase in [3H]dTTP incorporation, indicating that any effect of gallium on DNA polymerase alpha is minor. Gallium treatment did not induce or inhibit the repair of DNA single strand breaks. These data demonstrate that gallium inhibits replicative DNA synthesis, with the major specific enzyme target probably being ribonucleotide reductase.
Assuntos
Antineoplásicos/farmacologia , Replicação do DNA/efeitos dos fármacos , Gálio/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Desoxirribonucleotídeos/metabolismo , Desoxiuridina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Humanos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Trítio , Uridina/metabolismoRESUMO
Salvage of preformed nucleosides requires transport across the plasma membrane by sodium-dependent (concentrative) and sodium-independent (equilibrative) mechanisms. These transport systems are also the route of cellular uptake for nucleoside analogues, including gemcitabine (2',2'-difluorodeoxycytidine), a deoxycytidine analogue used in the treatment of pancreatic cancer. To determine whether gemcitabine cytotoxicity is influenced by the equilibrative-sensitive nucleoside transporter (es-NT), basal levels of the es-NT were quantified in three human pancreatic cancer cell lines (PANC-1, HS-766T, and PK-8) and one human bladder cancer cell line (MGH-U1) by flow cytometric analysis, and the results were compared with gemcitabine cytotoxicity assessed by clonogenic assay. To determine whether the salvage pathway of DNA synthesis can be up-regulated by inhibiting de novo DNA synthesis, combination experiments were carried out using the thymidylate synthase (TS) inhibitors 5-fluorouracil or raltitrexed with gemcitabine in a concurrent and sequential fashion. No relationship between basal es-NT and gemcitabine cytotoxicity was demonstrated. For two pancreatic cell lines, sequence-dependent effects of the combination of TS inhibitors and gemcitabine were seen with maximum effect when the TS inhibitors preceded gemcitabine. This was also associated with a significant increase in es-NT levels caused by the TS inhibitors. Thus, modulation of the es-NT by pretreatment with TS inhibitors may have the potential to improve the therapeutic benefit of gemcitabine.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Proteínas de Transporte/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/toxicidade , Antimetabólitos Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacocinética , Proteínas de Transporte de Nucleosídeo Equilibrativas , Citometria de Fluxo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Fluoruracila/administração & dosagem , Fluoruracila/toxicidade , Humanos , Concentração Inibidora 50 , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Nucleosídeos de Purina/metabolismo , Quinazolinas/administração & dosagem , Quinazolinas/toxicidade , Tiofenos/administração & dosagem , Tiofenos/toxicidade , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , GencitabinaRESUMO
The mitochondrial permeability transition and oxidative stress seem to be critical alterations in cellular physiology that take place during programmed cell death. Failure to undergo apoptosis is associated with drug resistance in acute myeloid leukemia and other cancers. Therefore, it is important to establish causal relationships between the physiological changes that take place in apoptosis, because these are potential targets for novel treatment strategies to overcome this form of drug resistance. We describe the use of multilaser flow cytometry methods to make correlated measurements of mitochondrial membrane potential (MMP), the generation of reactive oxygen intermediates, the cellular content of reduced glutathione (GSH), intracellular calcium, and exposure of phosphatidylserine on the cell surface. Using these combined methods, we have mapped a "death sequence" that occurs after treatment of leukemic blasts with clinically relevant concentrations of 1-beta-D-arabinofuranosylcytosine (ara-C). Dual labeling of MMP and cellular glutathione content showed that loss of MMP, indicative of the permeability transition, took place in cells that were depleted of glutathione. The loss of MMP coincided with phosphatidylserine exposure and preceded a state of high reactive oxygen generation. Finally, there was an increase in intracellular calcium. These results demonstrate that the mitochondrial permeability transition takes place during ara-C toxicity but suggest that this occurs downstream of the loss of GSH. Thus, oxidative stress after ara-C-induced toxicity seems to be a biphasic phenomenon, with the permeability transition occurring after a depletion of GSH and preceding a state of high reactive oxygen generation.
Assuntos
Citarabina/farmacologia , Mitocôndrias/fisiologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Cálcio/metabolismo , Citometria de Fluxo , Humanos , Membranas Intracelulares/fisiologia , Leucemia Mieloide Aguda , Potenciais da Membrana , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade , Fosfatidilserinas/metabolismo , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Using a novel flow cytometric method to analyze paraffin-embedded archival material, cellular DNA content of the primary tumor was measured in 165 patients with Stage II breast cancer who had been entered onto a large, multicenter trial of adjuvant chemotherapy. Fifty-three (32%) of the tumors examined were diploid, and the remainder contained one or more aneuploid clones. Aneuploid tumors had more extensive axillary lymph node involvement (p less than 0.05 using chi 2 analysis), but there were no significant correlations between cellular DNA content and either menopausal or estrogen receptor status. Forty-nine of 112 (44%) patients with aneuploid tumors have relapsed, compared to 12 of 53 (23%) with diploid tumors, and relapse-free survival curves show that beyond 2 years the diploid group reaches an apparent plateau, with a projected 4-year relapse-free survival of 72%, whereas the aneuploid group shows a continuing risk of relapse with only 43% remaining relapse free at 4 years. This correlation was most pronounced in the premenopausal patients; only 5 of 36 (14%) of those with diploid tumors have relapsed compared to 23 of 63 (37%) in the aneuploid group. However, multivariate analysis using a stepwise Cox model does not thus far confirm an independent prognostic significance of cellular DNA content on disease-free survival compared to node status. The cellular DNA content of the primary tumor did not appear to influence the patients' survival following relapse. These results indicate that compared to aneuploid tumors either diploid tumors have a different natural history or they are more responsive to adjuvant chemotherapy, possibly due to a lower probability of their developing drug resistance.
Assuntos
Neoplasias da Mama/patologia , DNA de Neoplasias/análise , Neoplasias da Mama/terapia , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Metástase Linfática , Menopausa , Estadiamento de Neoplasias , Ploidias , PrognósticoRESUMO
The hypoxia-inducible factor 1 (HIF-1) is known to induce the expression of several proteins linked to the maintenance of oxygen homeostasis, cellular energy metabolism, and tumor progression. Its alpha subunit (HIF-1alpha) is stabilized under hypoxic conditions and, therefore, might represent an intrinsic marker for tissue hypoxia. Here we report on the spatial relationship between HIF-1alpha and the nitroimidazole hypoxia marker EF5 in cervical carcinoma xenografts, and on their spatial relationship to tumor blood vessels. EF5 was administered to mice bearing ME180 and SiHa cervical cancer xenografts. Frozen tumor tissue sections, triple-stained for HIF-1alpha, the endothelial cell marker CD31, and EF5, were imaged using wide-field multiparameter immunofluorescence microscopy. Expression levels of EF5 and HIF-1alpha were similar in ME180 xenografts, but the percentage of tumor area stained with EF5 was significantly smaller than the percentage of HIF-1alpha-positive area in SiHa tumors. In both tumor types the EF5-HIF-1alpha overlap was statistically significant, thus confirming their spatial and temporal colocalization. Spatial distribution analysis of EF5 and HIF-1alpha is consistent with different pO2 value "thresholds" for EF5 binding and HIF-1alpha expression. Summarized, our results indicate that HIF-1alpha is a useful intrinsic marker for hypoxia in cervical carcinoma xenografts.
Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma de Células Escamosas/metabolismo , Etanidazol/análogos & derivados , Neoplasias do Colo do Útero/metabolismo , Animais , Carcinoma de Células Escamosas/irrigação sanguínea , Hipóxia Celular/fisiologia , Etanidazol/metabolismo , Feminino , Humanos , Hidrocarbonetos Fluorados/metabolismo , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Transplante de Neoplasias , Transplante Heterólogo , Neoplasias do Colo do Útero/irrigação sanguíneaRESUMO
Tumor cellular DNA content was measured by flow cytometry in 91 patients with advanced ovarian cancer, using a new and simple technique which allows archival paraffin-embedded tissue to be studied; 69% of the tumors were aneuploid, and 31% were diploid. Tumor ploidy was shown by multivariate Cox model analysis to be an independent prognostic variable and the major determinant of survival. Patients with diploid tumors survived significantly longer than did those with aneuploid tumors (p much less than 0.0001; X2 = 25.44; d.f. = 1).
Assuntos
DNA de Neoplasias/análise , Neoplasias Ovarianas/patologia , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Estadiamento de Neoplasias , Ploidias , PrognósticoRESUMO
The levels of expression of topoisomerase II alpha and topoisomerase II beta were investigated in six established cell lines of human childhood acute lymphoblastic leukemia (ALL) as a function of doubling time, cell cycle distribution, and of sensitivity to the antineoplastic agents Adriamycin and etoposide. The slowest growing cell line, ALL-G, was most sensitive to both drugs, whereas the fastest growing cell line, ALL-C, was 15.3- and 6.4-fold more resistant than ALL-G to Adriamycin and etoposide, respectively. Furthermore, ALL-W, the second most rapidly dividing cell line, was most resistant to both Adriamycin (22.8-fold) and etoposide (14.1-fold). Expression of topoisomerase II alpha varied inversely with doubling time, whereas no correlation was found between topoisomerase II beta levels and doubling time. Expression of topoisomerase II beta varied inversely with that of topoisomerase II alpha. The level of topoisomerase II alpha correlated directly with the percentage of cells in S and G2-M phases, whereas topoisomerase II beta expression varied directly with the number of cells in G1. An inverse correlation was found between the level of expression of topoisomerase II beta and resistance to Adriamycin, whereas a direct correlation was observed between the level of expression of topoisomerase II alpha and resistance to Adriamycin. Studies with etoposide, although not statistically significant, were consistent with the pattern observed with Adriamycin. These findings suggest that in ALL cells, cytocidal activity of Adriamycin and etoposide may be mediated, at least in part, by topoisomerase II beta.
Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Ciclo Celular , Divisão Celular , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Resistência a Medicamentos , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Células Tumorais CultivadasRESUMO
Bcl-2 is a key inhibitor of a broad range of apoptotic pathways, yet neither the mechanism of action nor the role of Bcl-2 subcellular localization are well understood. The subcellular localization of Bcl-2 includes the mitochondrial membrane as well as the contiguous membrane of the endoplasmic reticulum and nuclear envelope. Most studies suggest that the ability of Bcl-2 to confer cell survival is dependent upon its localization to the mitochondria. In this manuscript, we show that Bcl-2 targeted to the endoplasmic reticulum can inhibit Myc-, but not etoposide-induced apoptosis in the Rat-1 fibroblast cell line. By contrast, wild type Bcl-2 can inhibit apoptosis triggered by either death agonist. We further show both Myc and etoposide trigger disruption of mitochondrial membrane potential (MMP) and induce poly-ADP ribose polymerase (PARP) cleavage, but release of calcium was not evident. Bcl-2 abrogates apoptosis at or upstream of MMP depletion showing that Bcl-2 does not have to reside at the mitochondria to prevent apoptosis. These results further elucidate the biochemical events associated with Myc- and etoposide-induced apoptosis and significantly advance our understanding of Bcl-2 function.
Assuntos
Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspases/fisiologia , Linhagem Celular , Ativação Enzimática , Etoposídeo/farmacologia , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/fisiologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , RatosRESUMO
Since the over-expression of Bcl-2 is a common cause of multi-drug resistance, cytotoxic peptides that overcome the effects of Bcl-2 may be clinically useful. We harnessed the death-promoting alpha helical properties of the BH3 domain of BAD by fusing it to the Antennapedia (ANT) domain, which allows for cell entry (ANTBH3BAD). Treatment of 32D cells with the ANTBH3BAD peptide results in a 99% inhibition of colony formation. No significant toxicity is observed after treatment with ANT or BH3BAD alone. A mutant fusion peptide unable to bind Bcl-2 induces cell death as effectively as the wild-type ANTBH3BAD. Furthermore, 32D cells over-expressing Bcl-2 show no resistance to the ANTBH3BAD peptide. Therefore, the toxicity of the peptide was independent of the Bcl-2 pathway. We demonstrate that the toxicity of the peptide is due to its alpha helicity that disrupts mitochondrial function. Since this peptide overcomes major forms of drug resistance, it may be therapeutically useful if appropriately targeted to malignant cells.
Assuntos
Apoptose , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Fatores de Transcrição , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Proteína do Homeodomínio de Antennapedia , Proteínas de Transporte/genética , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Dicroísmo Circular , Ativação Enzimática , Feminino , Células HeLa , Proteínas de Homeodomínio/química , Humanos , Potenciais da Membrana , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Proteína de Morte Celular Associada a bclRESUMO
The prognostic value of cellular DNA content in ovarian cancer (malignant common epithelial tumors) was investigated by flow cytometric analysis of paraffin-embedded tumor blocks from 128 previously untreated patients with International Federation of Gynecology and Obstetrics (FIGO) stage III and IV ovarian cancer entered in a prospective clinical trial of combination v sequential therapy with chlorambucil and cisplatin. Seventy-three percent of tumors were aneuploid and 27% were diploid. Multivariate analysis using a Cox model showed that cellular DNA content (P less than .001) and FIGO stage (P less than .02) were the only significant independent prognostic variables. The median survival was 13 months for patients with aneuploid tumors and 60 months for patients with diploid tumors (P less than .0001). Further analysis indicated that the good prognosis associated with diploid tumors was limited to patients with stage III disease, all patients with stage IV (spread beyond the peritoneal cavity or liver metastases) disease having a poor prognosis irrespective of ploidy. On pathological review, nine borderline ovarian tumors (of low malignant potential) were identified, and seven of these were diploid. These tumors have an unusually favorable prognosis, despite apparent dissemination within the peritoneal cavity, a paradox which is often difficult to explain using conventional histological criteria. Although the vast majority of tumors in this study (93%) were classified as invasive epithelial ovarian cancers, it is possible that some of the patients with stage III diploid tumors may have had malignancies that were predominantly of low malignant potential, thus accounting in part for the prognostic significance of DNA content. By incorporating flow cytometric DNA analysis with careful histopathological assessment, it may be possible to better identify patients with an inherently good prognosis. This assumes particular importance, as the relatively favorable prognosis of patients with stage III diploid ovarian tumors appears to be independent of the type of treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , DNA de Neoplasias/análise , Citometria de Fluxo , Neoplasias Ovarianas/análise , Ensaios Clínicos como Assunto , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Ploidias , Prognóstico , Distribuição AleatóriaRESUMO
Cytosine arabinoside is usually considered to be lethal by incorporation into DNA followed by chain termination. Recently, we have reported that the radical scavenger N-acetyl-cysteine (NAC) protects cultured clonogenic AML blast cells from the lethal affects of Ara-C if given before the drug. This observation provides indirect evidence that toxic reactive oxygen intermediates (ROI) are generated in AML blast cells following Ara-C-induced damage to DNA. In the present paper we present evidence in support of this hypothesis. Using flow cytometry and multiple fluorescent probes for live cell function, we have mapped a sequence of discrete stages that occur during Ara-C cytotoxicity. An early event was the increased generation of ROI. Initially this oxidative stress was countered by an increase in the cellular content of reduced glutathione (GSH), but cells then underwent an abrupt transition to a state characterized by low GSH and very high ROI generation indicative of collapse of cellular redox balance. Next, the capacity to maintain low intracellular ionized calcium was lost, probably due to lipid peroxidation at membrane sites of calcium regulation. Finally, surface membrane integrity was lost. Concurrent measurements of clonogenic cell survival insured the relevance of these flow cytometry measurements to the stem cell population. We used OCI/AML-2 cells transfected with bcl-2 to look for the place in this sequence where bcl-2 protein protects cells against apoptosis; bcl-2 transfectants showed an increase in ROI generation similar to controls, but were able to maintain GSH levels in the face of this oxidative stress. We conclude that oxidative stress plays a major role in Ara-C toxicity, and that bcl-2 protein protects cells by maintaining cellular redox balance in a reducing state. These studies complement previous work showing how regulators of AML growth affect the sensitivity of blast cells to Ara-C by changing the concentration or stability of bcl-2 protein.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Crise Blástica/patologia , Citarabina/farmacologia , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Crise Blástica/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Citometria de Fluxo , Glutationa/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Peroxidação de Lipídeos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Oxirredução , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2 , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Ensaio Tumoral de Célula-TroncoRESUMO
Expression of the multidrug resistance efflux pump P-glycoprotein (Pgp) was measured in a series of AML patients using two flow cytometry methods. Transport function was assessed by measuring the modulating effect of the Pgp inhibitor cyclosporin A (CsA) on the cellular accumulation of daunorubicin, and Pgp antigen expression by surface immunofluorescence using the MRK-16 antibody. Both methods showed a wide range of values for Pgp expression between individual patients, but in contrast to a series of cell lines expressing Pgp there was no correlation between antigen expression and transport function in the clinical samples. As previously reported for chronic lymphocytic leukemia (CLL), pretreatment with neuraminidase markedly improved MRK-16 staining in some cases, indicating that abnormal glycosylation can cause epitope masking in AML blasts. Because experience with cell lines shows that Pgp expression is a continuous variable which correlates with the level of drug resistance, rather than the 'positive' or 'negative' which are frequently reported by clinical flow cytometry laboratories, we used a calibration procedure to estimate the actual number of Pgp molecules expressed in the AML samples. Despite the additional refinements of neuraminidase treatment and antigen quantification, the correlation between Pgp antigen expression and daunorubicin accumulation remained extremely weak (r = 0.11; P = 0.63). It is suggested that the assay for transport function can detect molecules that affect daunorubicin accumulation but are antigenically distinct from classical P-glycoprotein. Heterogeneity of multidrug resistance efflux pumps might in part explain the relatively weak prognostic significance of immunofluorescence detection of Pgp in AML patients.