RESUMEN
CD34+ cell dose calculations are usually based on actual body weight (ABW). We have shown that ideal body weight (IBW) may provide a better basis for this in a small population of patients with hematologic malignancies. This was studied further in 514 myeloma autografts. The CD34+ cell doses (10(6)/kg) by IBW and ABW were 1.37-39.36 (median 6.03) and 1.15-29.67 (median 4.84), respectively. IBW-based cell doses correlated slightly better with engraftment than ABW-based doses (higher r(2)): 0.5 x 10(9)/l neutrophils 0.83 versus 0.82, 1.0 x 10(9)/l neutrophils 0.78 versus 0.77, 20 x 10(9)/l platelets 0.54 versus 0.53 and 50 x 10(9)/l platelets 0.57 versus 0.55. When outliers (hematologic recovery in <8 or >16 days) were excluded, the findings were similar: 0.5 x 10(9)/l neutrophils 0.85 versus 0.84, 1.0 x 10(9)/l neutrophils 0.85 versus 0.84, 20 x 10(9)/l platelets 0.86 versus 0.85 and 50 x 10(9)/l platelets 0.85 versus 0.84. CD34+ cell doses based on IBW as well as ABW significantly affected engraftment when analyzed separately as continuous variables. However, when analyzed together, only the dose based on IBW retained significance. We conclude that calculation of CD34+ cell numbers for autotransplantation should be based on IBW.
Asunto(s)
Antígenos CD34/sangre , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Mieloma Múltiple/terapia , Adulto , Anciano , Biomarcadores/sangre , Índice de Masa Corporal , Peso Corporal , Terapia Combinada , Femenino , Neoplasias Hematológicas/sangre , Movilización de Célula Madre Hematopoyética/métodos , Humanos , Masculino , Melfalán/uso terapéutico , Persona de Mediana Edad , Mieloma Múltiple/tratamiento farmacológico , Estudios Retrospectivos , Trasplante Autólogo/métodosRESUMEN
Recent advances in purine analog-based combination chemotherapy and chemoimmunotherapy have significantly improved response rates and progression-free survival in patients with B-cell chronic lymphocytic leukemia (CLL). However, there are clinical scenarios in which purine analog-based treatment may not be appropriate, either because of the risk of toxicity in patients with comorbidity or because purine analog-based therapies are unlikely to achieve satisfactory responses. Novel, nonchemotherapeutic treatment regimens are becoming increasingly important in these patients, as well as in patients in whom combination chemotherapy-based treatment has failed or resulted in relapse. Nonchemotherapeutic agents include monoclonal antibodies, glucocorticoids, immunomodulatory drugs, drugs with specific intracellular molecular targets, vaccines and cellular immunotherapies. These agents use diverse mechanisms of action that may complement each other, therefore providing a scientific rationale to investigate combinations of these agents in the treatment of CLL. In this review, we will discuss current knowledge of available nonchemotherapeutic agents, available clinical experience with their use alone and in combination and how these approaches may affect outcomes in patients with CLL.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunoterapia , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Anciano , Anticuerpos Monoclonales/efectos adversos , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/uso terapéutico , Terapia Combinada , Sistemas de Liberación de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Glucocorticoides/efectos adversos , Glucocorticoides/uso terapéutico , Humanos , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/uso terapéutico , Inmunosupresores/efectos adversos , Inmunosupresores/uso terapéutico , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Inmunoterapia Activa/efectos adversos , Inmunoterapia Adoptiva/efectos adversos , Persona de Mediana Edad , Ensayos Clínicos Controlados Aleatorios como Asunto/estadística & datos numéricosRESUMEN
Seventy-one allograft recipients receiving voriconazole, in whom complete clinical, microbiologic and pharmacokinetic data were available, were studied to determine the efficacy of voriconazole in preventing fungal infections. The length of voriconazole therapy was 6-956 days (median 133). The total number of patient-days on voriconazole was 13 805 ( approximately 38 years). A total of 10 fungal infections were seen in patients on voriconazole (18% actuarial probability at 1 year): Candida glabrata (n=5), Candida krusei (n=1), Cunninghamella (n=1), Rhizopus (n=2) and Mucor (n=1). Two of the four zygomycosis cases were preceded by short durations of voriconazole therapy, but prolonged itraconazole prophylaxis. The plasma steady-state trough voriconazole levels around the time the infection occurred were <0.2, <0.2, 0.33, 0.55, 0.63 and 1.78 microg/ml in the six candidiasis cases. Excluding the four zygomycosis cases, all the six candidiasis cases were seen among the 43 patients with voriconazole levels of < or =2 microg/ml and none among the 24 with levels of >2 microg/ml (P=0.061). We conclude that voriconazole is effective at preventing aspergillosis. However, breakthrough zygomycosis is seen in a small proportion of patients. The role of therapeutic voriconazole monitoring with dose adjustment to avoid breakthrough infections with fungi that are otherwise susceptible to the drug needs to be explored prospectively.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/efectos adversos , Micosis/tratamiento farmacológico , Premedicación , Pirimidinas/uso terapéutico , Triazoles/uso terapéutico , Antifúngicos/uso terapéutico , Aspergilosis/tratamiento farmacológico , Aspergilosis/prevención & control , Candidiasis/tratamiento farmacológico , Recolección de Datos , Evaluación de Medicamentos , Femenino , Humanos , Masculino , Pirimidinas/sangre , Pirimidinas/farmacocinética , Trasplante Homólogo , Triazoles/sangre , Triazoles/farmacocinética , Voriconazol , Cigomicosis/tratamiento farmacológicoAsunto(s)
Pérdida Auditiva Sensorineural/etiología , Hemorragia , Enfermedades del Laberinto/complicaciones , Cóclea/patología , Femenino , Pérdida Auditiva Sensorineural/patología , Humanos , Enfermedades del Laberinto/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/complicaciones , Imagen por Resonancia Magnética/métodos , Persona de Mediana Edad , Vestíbulo del Laberinto/patologíaRESUMEN
Sixty three patients aged 27-66 years (median 52) were allografted from HLA-matched sibling (n=47), 10 of 10 allele-matched unrelated (n=19), or one-antigen/allele-mismatched (n=7) donors aged 24-69 years (median 46) after a conditioning regimen comprising 100 mg/m(2) melphalan. Cyclophosphamide (50 mg/kg) was also administered to patients who had not been autografted previously. Cyclosporine or tacrolimus, and mycophenolate mofetil were administered to prevent graft-versus-host disease (GVHD). The 2-year cumulative incidences of relapse and TRM were 55 and 24% respectively, and 2-year probabilities of overall survival (OS) and disease-free survival (DFS) were 36 and 21%, respectively. Poor performance status, donor age >45 years and elevated lactate dehydrogenase (LDH) increased the risk of treatment-related mortality (TRM), refractory disease and donor age >45 years increased the risk of relapse, and OS and DFS were adversely influenced by refractory disease, poor performance status, increased LDH, and donor age >45 years. Our data suggest that younger donor age is associated with better outcome after sub-myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) for hematologic malignancies due to lower TRM and relapse. This finding raises the question of whether a young 10-allele-matched unrelated donor is superior to an older matched sibling donor in patients where the clinical situation permits a choice between such donors.
Asunto(s)
Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Adulto , Factores de Edad , Anciano , Análisis de Varianza , Supervivencia sin Enfermedad , Femenino , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/complicaciones , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , Recurrencia , Estudios Retrospectivos , Factores de Riesgo , Hermanos , Tasa de Supervivencia , Trasplante Homólogo , Resultado del TratamientoRESUMEN
Whether the CD34+ and CD3+ cell doses in allogeneic HSCT should be estimated using actual (ABW) or ideal (IBW) body weight has never been definitively determined. We have shown that CD34+ cell doses based upon IBW are better predictive of engraftment after autologous and allogeneic HSCT. Sixty-three patients undergoing reduced-intensity HSCT after a uniform preparative regimen were evaluated to determine the effect of cell dose. ABW and IBW were 45-147 kg (median 79) and 52-85 kg (median 67) respectively. The ABW-IBW difference was -24% to +133% (median +16%); nine patients were >5% underweight and 41 were >5% overweight. The CD34+ cell dose (10(6)/kg) was 1.4-11.8 (median 5) by IBW and 1.2-9.3 (median 4.5) by ABW. The CD3+ cell dose (10(8)/kg) was 0.9-14.9 (median 3) by IBW and 0.7-19.7 (median 2.7) by ABW. While CD34+ and CD3+ cell doses based upon IBW were found to affect transplant-related mortality, and disease-free and overall survival significantly, those based on ABW were either not predictive of outcome or the differences were of borderline significance. We suggest using IBW rather than ABW to calculate cell doses for HSCT; for statistical analyses and for clinical practice if a specific cell dose is being targeted.
Asunto(s)
Peso Corporal , Trasplante de Células Madre/métodos , Adulto , Anciano , Antígenos CD/análisis , Antígenos CD34/análisis , Recuento de Células , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sobrepeso , Trasplante de Células Madre/mortalidad , Análisis de Supervivencia , Delgadez , Recolección de Tejidos y Órganos/métodos , Trasplante Homólogo , Resultado del TratamientoRESUMEN
We have developed a solid-phase ELISA for the specific and sensitive detection of apoptotic cells. This method is based on the ability of a monoclonal antibody (MAb) against single-stranded DNA (ssDNA) to specifically identify apoptotic cells. The assay involves binding of cells to 96-well microtiter plates, treatment of the attached cells with formamide to denature DNA in apoptotic cells and one-step staining of the denatured DNA with a mixture of anti-ssDNA MAb and peroxidase-conjugated anti-mouse IgM. A near linear increase in signal was seen as the number of apoptotic cells increased from 500 to 5000. Untreated and necrotic cells or cells with single-stranded DNA breaks induced by H(2)O(2) did not produce signal above the background level. In leukemic cell cultures grown, treated with ID(50) concentration of etoposide, stained and analyzed in the same 96-well assay plate, intense ELISA signal was detected. The ratio of absorbance values from drug resistant and drug-sensitive cell lines treated with etoposide was in agreement with the degree of resistance determined by growth inhibition assays. These data show that this ELISA has sufficient sensitivity for use in drug screening protocols. In breast cancer cell cultures treated with cisplatin, ELISA absorbance increased only after treatment with drug concentrations 10-fold higher than concentrations inducing 95% growth inhibition. In cultures treated with staurosporine, there was a near linear relation between the ELISA absorbance values and cytotoxicity in the range of 15-92% growth inhibition. The absence of apoptotic signal in breast cancer cells treated with cytotoxic concentrations of cisplatin indicated that this drug kills cells by non-apoptotic mechanisms, whereas apoptosis was the dominant mechanism of cell death caused by staurosporine. The formamide-MAb apoptosis ELISA described here may provide a basis for high-throughput screening of drugs based on their ability to induce or suppress apoptosis.
Asunto(s)
Apoptosis , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos Monoclonales/inmunología , Antineoplásicos/farmacología , División Celular , ADN de Cadena Simple/inmunología , Evaluación Preclínica de Medicamentos/métodos , Formamidas/química , Humanos , Neoplasias/patología , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide-MAb assay for the apoptotic cells. However, TUNEL stained 90-100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA-histone interactions. Importantly, the formamide-MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues.
Asunto(s)
Apoptosis , Cromatina/metabolismo , ADN/metabolismo , Formamidas/farmacología , Animales , Anticuerpos Monoclonales , Inhibidores de Caspasas , Cromatina/química , ADN/química , ADN/inmunología , ADN de Cadena Simple/química , ADN de Cadena Simple/inmunología , ADN de Cadena Simple/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Fijadores , Citometría de Flujo , Formaldehído , Secciones por Congelación , Humanos , Etiquetado Corte-Fin in Situ , Indicadores y Reactivos , Cinética , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Necrosis , Desnaturalización de Ácido Nucleico , Adhesión en Parafina , Temperatura , Células Tumorales CultivadasRESUMEN
Precise quantitation of apoptotic cells in solid tumors is necessary to determine the role of apoptosis in cancer growth, prognosis, and treatment. In this study, the intensity of apoptotic death was determined in 91 breast carcinomas with a novel cellular marker of apoptosis based on the staining of histological sections with a monoclonal antibody (MAb) to single-stranded DNA. Staining of apoptotic cells with the MAb reflected the decreased thermal stability of DNA induced by the digestion of nuclear proteins, as demonstrated by the elimination of staining in sections reconstituted with histones before heating. The high sensitivity and specificity of apoptosis analysis with the MAb is based on the central role of protease activation in the mechanism and control of apoptosis. Apoptotic indexes (AIs) in breast carcinomas ranged between 0 and 46%. Most of the carcinomas had relatively low AIs, whereas 29 cases were classified as carcinomas with intensive apoptosis (AI >/= 10%). The high level of apoptotic cell death was associated with negative immunostaining for bcl-2 protein, the loss of estrogen and progesterone receptors, high proportion of cells in S-phase, and increased risk of lymph node metastases. There was no correlation between AI and tumor size or p53 immunostaining. Lymph node metastases were detected in 59% of patients with high levels of apoptosis in primary carcinomas and in only 21% of patients with AIs below 10% in primary carcinomas. Thus, the high sensitivity of the MAb assay made it possible to identify a subset of breast carcinomas with intensive apoptosis and markers of poor prognosis. These results demonstrate that the measurement of apoptosis in breast carcinomas provides valuable prognostic information.
Asunto(s)
Apoptosis , Neoplasias de la Mama/patología , ADN de Neoplasias/análisis , ADN de Cadena Simple/análisis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/cirugía , Femenino , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Neoplasias Pulmonares/patología , Metástasis Linfática , Linfoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Fase S , Proteína p53 Supresora de Tumor/análisisRESUMEN
Chronic lymphocytic leukemia (CLL) cells were cultured in a medium supplemented with 0.01-1 ng/ml interleukin-4 (IL-4) for 18 h, fixed and analyzed on a flow cytometer. The percentage of apoptotic (AP) cells with hypodiploid DNA content was determined from DNA histograms. IL-4 at 0.01 ng/ml protected from spontaneous apoptosis of cells from previously treated CLL patients, but had very little effect on apoptotic death in cultures of cells from untreated patients. The number of AP cells in the absence of IL-4 was similar in cultures from treated and untreated patients. The concentration of IL-4 which inhibited spontaneous apoptosis by 50% was less than 0.01 ng/ml for pretreated patients and close to 1 ng/ml for untreated patients. Stage of the disease had no effect on the level of spontaneous apoptosis and its sensitivity to IL-4. Protection from apoptosis by IL-4 was not accompanied by the upregulation of bcl-2 protein. The number of AP cells in methylprednisolone hemisuccinate (MP) treated cultures from previously treated patients was significantly lower than in cultures from untreated patients in the presence of 0.01-1.0 ng/ml IL-4. Treatment with the combination L-phenylalanine mustard (L-PAM)+ fludarabine induced synergistic apoptotic response. Apoptosis induced by this combination was relatively resistant to IL-4 in patients treated with chlorambucil and prednisone, but not in patients previously treated with fludarabine. Protection from cytotoxicity by IL-4 may be one of the mechanisms of acquired drug resistance in CLL.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Interleucina-4/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Sinergismo Farmacológico , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Linfocitos/efectos de los fármacos , Masculino , Melfalán/farmacología , Persona de Mediana Edad , Células Tumorales Cultivadas/efectos de los fármacos , Vidarabina/análogos & derivados , Vidarabina/farmacologíaRESUMEN
The most widely used histochemical marker of apoptosis (in situ end labeling, TUNEL) detects both apoptotic and necrotic cells and evaluates only late stages of apoptosis. Hence, a specific and sensitive cellular marker of apoptosis is needed to determine the role of apoptotic death in biology and pathology. The present study describes a novel immunohistochemical procedure for the staining of apoptotic cells using a monoclonal antibody (MAb) to single-stranded DNA. This MAb stained all cells with the morphology typical of apoptosis in etoposide-treated HL-60, MOLT-4, and R9 cell cultures, in which apoptosis was accompanied by high, moderate, and low levels of internucleosomal DNA fragmentation, respectively. TUNEL stained all apoptotic cells in HL-60 cultures, nearly 60% of apoptotic cells in MOLT-4 cultures, and only 14% of apoptotic cells in R9 cultures. Apoptotic R9 cells, which progressed into secondary necrosis, retained MAb staining and became TUNEL-positive. Necrotic cells in MOLT-4 cultures treated with sodium azide were stained by TUNEL, but were negative for MAb staining. All floating cells at a late stage of apoptosis in MDA-MB-468 cultures treated with cisplatin were stained by both MAb and TUNEL. However, among adherent cells in the early stages of apoptosis, MAb stained nearly 20 times more cells than TUNEL. In histological sections of human tumor xenografts, MAb detected clusters of apoptotic cells in viable tumor tissue, but did not stain cells in areas of central ischemic necrosis. In contrast, TUNEL stained nuclei in necrotic areas. Thus, MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis.
Asunto(s)
Anticuerpos Monoclonales , Apoptosis , ADN de Cadena Simple/análisis , Técnica del Anticuerpo Fluorescente Indirecta , Inmunohistoquímica , Animales , Biomarcadores , Línea Celular , Cisplatino/farmacología , ADN de Cadena Simple/inmunología , Etopósido/farmacología , Células HL-60 , Humanos , Leucemia/patología , Ratones , Necrosis , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
We report application of a novel immunohistochemical procedure for the staining of apoptotic (AP) cells in paraffin sections using monoclonal antibody (MAb) to single-stranded DNA. MAb differentiated between apoptosis and necrosis and in contrast to in situ end labelling specifically stained only AP cells. AP carcinoma cells stained with the antibody were detected in 32 of 58 infiltrating human breast carcinomas and in 9 of 15 colon adenocarcinomas. Stromal cells stained with the MAb were observed in all carcinomas, including those in which no AP carcinoma cells were detected. There was a strong positive correlation between the presence of AP cells, loss of hormone receptors and a high proliferation rate in breast carcinomas. AP cells were present in 80-87% of receptor-negative carcinomas, while most of receptor-positive breast carcinomas did not contain AP cells. Apoptosis in tumor cells was detected significantly more frequently among breast carcinomas with high, than among carcinomas with low S-phase fraction. AP cells were present in 93-95% of breast carcinomas which were receptor-negative and had a high S-phase fraction. Immunostaining demonstrated a strong positive correlation between the loss of bcl-2 protein and intensive apoptosis in breast carcinomas. Association between apoptosis and markers of poor prognosis in breast cancer (loss of hormone receptors, intensive proliferation, loss of bcl-2 protein) indicates that apoptotic cell death is typical of more aggressive carcinomas.
Asunto(s)
Adenocarcinoma/patología , Apoptosis , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , ADN de Neoplasias/análisis , ADN de Cadena Simple/análisis , Neoplasias Gastrointestinales/patología , Anticuerpos Monoclonales , Biopsia , Neoplasias de la Mama/cirugía , Neoplasias del Colon/patología , Femenino , Neoplasias Gastrointestinales/cirugía , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Necrosis , Neoplasias Gástricas/patología , Células Tumorales CultivadasRESUMEN
Breast cancer cells are relatively resistant to the induction of apoptosis (AP) and drug regimens which readily activate apoptotic death, may enhance the antitumor effect. Rapid and intensive induction of apoptosis was observed in estrogen receptor positive and negative breast cancer cell cultures treated with tamoxifen (TMX) combined with the calmodulin antagonists trifluoperazine (TFP) or W7. TMX (1-5 microM) alone or calmodulin antagonists alone did not induce apoptosis. Importantly, intensive apoptosis was also induced by TMX and TFP in the cells obtained from primary human breast carcinomas. Inhibition of the Ca2+ calmodulin signaling pathway is an effective way to activate apoptotic death in epithelial cells. Combination of TMX with non-toxic calmodulin inhibitors may increase the preventive and therapeutic effects of TMX.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Calmodulina/antagonistas & inhibidores , Antagonistas de Estrógenos/farmacología , Tamoxifeno/farmacología , Trifluoperazina/farmacología , Sinergismo Farmacológico , Femenino , Humanos , Células Tumorales CultivadasRESUMEN
Cell line R9 generated by continuous exposure of MOLT-4 cells to adriamycin was cross-resistant to a variety of unrelated drugs. The following data indicate that diminished apoptotic response was the mechanism of acquired pleiotropic drug resistance: (i) apoptosis was a common mechanism of cell death for agents expressing cross-resistance; (ii) induction of apoptosis by drugs, medium depletion and serum deprivation was decreased in R9 cells; (iii) DNA degradation in apoptotic cells was lower in resistant lines, probably reflecting a modification of apoptotic pathway in resistant cells; (iv) inhibition of cell division and DNA synthesis by drugs was similar in sensitive and resistant cells. These data indicated a similar level of initial damage, as typical for resistance based on modified apoptotic response. There was no difference in bcl-2 protein level between sensitive and resistant cells. Thus acquired pleiotropic resistance and diminished apoptotic response in R9 cells were induced by a bcl-2-independent mechanism. Surface T-cell antigen CD4 was expressed in MOLT-4 and lost in R9 cells. The role of CD4 down-regulation in apoptosis-related drug resistance remains to be explored. The association between acquired pleiotropic drug resistance and increased survival capacity in unfavorable growth conditions indicated that drug-induced selection of cells with diminished apoptotic response may stimulate neoplastic progression. Alkylating agents induced similar cytotoxicity and only slightly lower apoptosis in R9 cells in comparison with MOLT-4 cells. Our data show that some drugs may overcome acquired pleiotropic drug resistance based on the modified apoptotic pathway.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Leucemia Linfoide/tratamiento farmacológico , Leucemia Linfoide/patología , División Celular/fisiología , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Humanos , Cinética , Leucemia Linfoide/metabolismo , Microscopía Fluorescente , Fenotipo , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2 , Rodamina 123 , Rodaminas/farmacocinéticaRESUMEN
In cultures of leukemic HL-60 and MOLT-4 cells treated with etoposide all nuclei with distinctive morphology of apoptosis (chromatin condensation at the nuclear periphery, nuclear fragmentation) were stained with monoclonal antibody F7-26 specific for single-stranded DNA. DNA in interphase and mitotic cells of control cultures and DNA in necrotic cells of cultures treated with sodium azide did not bind the antibody. In monolayer cultures of breast cancer cell line MDA-468 treated with tamoxifen for 4 hours all cells detached from substratum. These cells were apoptotic by nuclear morphology and stained with F7-26. Subset of cells without visible chromatin condensation but stained with F7-26 was detected among cells still attached to the substratum 1 hour after addition of tamoxifen. Thus, in breast cancer cells reactivity with F7-26 preceded chromatin condensation detected by fluorescence microscopy. Apoptotic cells stained with the antibody and non-apoptotic cells with background fluorescence were completely separated on two-parameter plots generated on a flow cytometer. Linear relation between percentage of apoptotic cells and ELISA reactivity with F7-26 in the cells attached to microtiter plates was demonstrated. These data show that apoptotic response can be measured by ELISA using staining with F7-26. Cells undergoing apoptosis can be detected by the procedure based on thermal denaturation of DNA in situ in the presence of Mg2+ with subsequent staining with the antibody specific for DNA in single-stranded conformation. Correlation between nuclear morphology typical of apoptosis in various cell types demonstrated that staining with monoclonal antibody F7-26 provides specific cytochemical marker for apoptotic cells.
Asunto(s)
Anticuerpos Monoclonales , Apoptosis/fisiología , Neoplasias de la Mama/patología , ADN de Neoplasias/análisis , ADN de Cadena Simple/análisis , Leucemia Promielocítica Aguda/patología , Leucemia de Células T/patología , Animales , Anticuerpos Monoclonales/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Adhesión Celular/fisiología , Núcleo Celular/química , Cromatina/química , ADN de Neoplasias/inmunología , ADN de Neoplasias/metabolismo , ADN de Cadena Simple/inmunología , ADN de Cadena Simple/metabolismo , Ensayo de Inmunoadsorción Enzimática , Etopósido/farmacología , Citometría de Flujo , Fluorescencia , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Leucemia de Células T/tratamiento farmacológico , Leucemia de Células T/metabolismo , Magnesio/farmacología , Ratones , Ratones Endogámicos BALB C , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Coloración y Etiquetado , Células Tumorales CultivadasRESUMEN
To determine the relation between apoptosis and cytotoxicity the number of apoptotic (AP) cells was measured in MOLT-4 and HL-60 cultures treated with adriamycin, etoposide, cisplatin and 1-B-D-arabinofuranosylcytosine (ara-C) in the dose range inducing 20-95% growth inhibition. DNA degradation in individual AP cells was identified with anti-ssDNA monoclonal antibody. There was a near linear relationship between the number of AP cells and drug dose. All drugs induced apoptosis at a comparable level of cytotoxicity. Higher chemosensitivity of MOLT-4 than HL-60 cells was in agreement with the higher number of AP cells. Synergistic cytotoxicity of drug combinations was predicted by apoptosis assay. The number of AP cells was a reliable indicator of chemosensitivity when various cell lines and different drugs were compared. Low doses of cyclohexemide (CHX) inhibited etoposide-induced apoptosis when cells were exposed to both drugs for 6 h. Treatment with CHX alone during longer period or at higher doses induced apoptosis. Inhibition or induction of apoptosis by CHX in the same cell line was determined by the dose of drugs and the time of treatment. MOLT-4/Adr subline developed by continuous exposure to adriamycin was 1.6 - 2.3 fold resistant to adriamycin, etoposide and ara-C by growth inhibition and apoptosis assays. Spontaneous apoptosis induced by medium depletion was decreased in resistant cells. The selection of cells with diminished apoptotic response at early stage of acquired resistance induced pleoitropic drug resistance.
RESUMEN
Aphidicolin (AP) or hydroxyurea (HU) inhibited DNA repair and enhanced cytotoxicity in human ovarian carcinoma cells A2780 treated with L-phenylalanine mustard (L-PAM) combined with cisplatin or thioTEPA, and in the cells treated with cisplatin combined with thioTEPA. In cultures treated with L-PAM or cisplatin alone post-treatment with AP or HU had no effect on DNA repair and produced only additive cytotoxicity. Post-treatment with AP + HU inhibited DNA repair and enhanced cell killing in cultures treated with L-PAM alone. The inhibitor of protein synthesis cycloheximide protected cells from the cytotoxicity of AP + HU but had no effect on synergistic cell killing produced by DNA repair inhibition. In cisplatin-resistant cells A2780/CP post-treatment with AP + HU enhanced the cytotoxicity of L-PAM, but not of cisplatin. However, in resistant cells treated with cisplatin combined with L-PAM or thioTEPA DNA repair inhibitors decreased IC90 of cisplatin. Treatment of cells with two alkylating agents enhanced the sensitivity to DNA repair inhibitors and eliminated low sensitivity to inhibitors of repair associated with drug resistance.
Asunto(s)
Alquilantes/toxicidad , Antineoplásicos/toxicidad , Supervivencia Celular/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Afidicolina/toxicidad , Cisplatino/toxicidad , Citarabina/toxicidad , Interacciones Farmacológicas , Femenino , Humanos , Hidroxiurea/toxicidad , Melfalán/toxicidad , Neoplasias Ováricas , Tiotepa/toxicidad , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
Chronic lymphocytic leukemia and lymphoma cells were treated with antitumor drugs in vitro and analyzed by flow cytometry to measure the number of apoptotic (AP) cells and DNA damage in the cells that escaped apoptotic death. AP cells were identified by a high sensitivity of DNA to thermal denaturation, which induced binding of antibody to single-stranded DNA, and by decreased stainability of cells with the intercalating DNA dye propidium iodide. The appearance of AP cells was prevented by Zn++ and inhibited by phorbol ester. AP cells were induced by alkylating agents, antimetabolites, and anthracyclines. A linear relationship between L-phenylalanine mustard dose and the number of AP cells was observed. A synergistic interaction between drugs was detected by an increased number of AP cells and by the intensity of DNA damage in non-apoptotic cells. A most interesting example of synergism was the combination of alkylating agents with fludarabine. Linearity of dose-response curves, and the capability to detect drug synergism and to evaluate variable response of cells from different patients to single agents and combinations suggest that flow cytometry of apoptosis will provide a basis for chemosensitivity tests in leukemia and lymphoma.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Linfoma/tratamiento farmacológico , Linfoma/patología , Daño del ADN , Ensayos de Selección de Medicamentos Antitumorales/métodos , Citometría de Flujo , Humanos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
DNA damage was evaluated by flow cytometric (FCM) analysis of cells treated with L-phenylalanine mustard (L-PAM) and stained with anti-DNA monoclonal antibody (MAb) F7-26. DNA damage was rapidly repaired, as indicated by the loss of DNA immunoreactivity after removal of L-PAM. Two types of drug combinations were found to inhibit DNA repair. Combinations containing inhibitors of DNA polymerase (ara-C, aphidicolin) or these inhibitors and hydroxyurea inhibited DNA repair in A2780/PAM and A549 cells. The inhibition of DNA repair by combinations of DNA-damaging agents thioTEPA or cisplatin and DNA polymerase inhibitors is a novel observation based on the specificity of DNA damage assay with MAb F7-26. Combinations containing thioTEPA or cisplatin inhibited DNA repair in A549 but not in A2780/PAM cells. Drug combinations which inhibited DNA repair also significantly enhanced cell killing by L-PAM. Cell survival in cultures treated with L-PAM and efficient inhibitors was 2 to 3 orders of magnitude lower than was expected for additive survival. ThioTEPA and cisplatin play a dual role in combination chemotherapy by inducing DNA damage and inhibiting repair of DNA damage. FCM analysis of DNA repair may be a useful component of drug evaluation and could be applied to determine cell-type specific sensitivity to inhibitors of DNA repair.