RESUMEN

We used human tumor cell lines from the National Cancer Institute's In Vitro Antineoplastic Drug Screen to assess whether sensitivity to any of the approximately 45,000 compounds tested previously correlated with the presence of a ras oncogene. Among these cell lines, the mutations in Ki-ras2 clustered in non-small cell lung and colon carcinoma subpanels, and five of the six leukemia lines contained mutations in either N-ras or Ki-ras2. These analyses revealed a striking correlation with 1-beta-D-arabinofuranosylcytosine (Ara-C) and 2,2'-O-cyclocytidine sensitivity in the cell lines harboring ras mutations compared to the tumor lines with wild-type ras alleles. Strong correlations were also found with topoisomerase (topo) II inhibitors, especially 3'-hydroxydaunorubicin and an olivacine derivative. These differential sensitivities persisted in an additional 22 non-small cell lung carcinoma lines (ras mutations, n = 12 and wild-type ras, n = 10). Thus, the association with Ara-C sensitivity was greatest while topo II inhibitors showed a lower, but significant, correlation. These results suggest that the ras oncogene may play a determinant role in rendering tumor cells sensitive to deoxycytidine analogues and topo II inhibitors.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Citarabina/farmacología , Genes ras/genética , Inhibidores de Topoisomerasa II , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Citarabina/administración & dosificación , Análisis Mutacional de ADN , Daunorrubicina/administración & dosificación , Daunorrubicina/farmacología , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Programas Informáticos , Células Tumorales Cultivadas , Gemcitabina

RESUMEN

More than 200 human small cell lung cancer and non-small cell lung cancer cell lines were established over 15 years mainly by utilizing the serum-free, hormone and growth factor supplemented, defined media HITES and ACL4. Use of modified, established cell culture techniques such as the mechanical spillout method for the releasing of cell aggregates from tumor tissue, ficoll gradient centrifugation for the separation of tumor cells from erythrocytes and tissue debris, and an apparatue consisting of a platinum tubing attached to a suction flask for removal of spent medium have greatly contributed to the success in culturing tumor cells. Characterization of these lung cancer cell lines have extended our knowledge of lung cell biology. Studies elucidating the nutritional requirements of lung cancer cell growth may be helpful for the manipulation of these tumors in patients.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Pequeñas/patología , Técnicas de Cultivo de Célula/métodos , Neoplasias Pulmonares/patología , Ascitis/patología , Médula Ósea/patología , Técnicas de Cultivo de Célula/instrumentación , Medio de Cultivo Libre de Suero , Humanos , National Institutes of Health (U.S.) , Derrame Pleural/patología , Manejo de Especímenes , Células Tumorales Cultivadas , Estados Unidos

RESUMEN

Clinical protocols for small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) were devised to prospectively select individualized chemotherapy based on in vitro drug sensitivity testing (DST) of cell lines derived from the patient's SCLC tumor cell lines or the patient's fresh NSCLC tumor. DST data derived from SCLC tumor cell lines were available for 33/115 (29%) patients. The DST-selected chemotherapy regimen was administered to 21 (18%) patients, or 64% of patients with DST. In SCLC< the DST-selected chemotherapy was administered either during weeks 13-24 following 12 weeks of etoposide/cisplatin, or at relapse after complete response to etoposide/cisplatin. Several parameters of in vitro drug sensitivity were significantly associated (two-sided P < 0.05) with clinical response to primary therapy and also with response to the DST-selected chemotherapy regimen, but were not associated with survival (P = 0.24). Five patients treated with their DST-selected chemotherapy attained a complete or partial response, compared to 5 of 68 who received an empiric regimen (P = 0.057). A total of 36/165 (22%) NSCLC patients had DST successfully completed. These results directed management for 21/96 (22%) patients who eventually received chemotherapy, or 58% of patients with DST. Response to chemotherapy for the patients treated prospectively with their DST-selected chemotherapy regimen (2/21; 9%) was not significantly different than the response rate for patients treated empirically with etoposide/cisplatin (10/69; 14%) in the absence of in vitro results to direct chemotherapy (P = 0.73). There was no difference in survival by treatment group for the NSCLC patients. The correlation between in vitro and clinical response was not significant for any individual drug or for all drugs considered together, illustrating the poor predictive value of in vitro testing with currently available chemotherapy in NSCLC.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Pulmonares/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Carcinoma de Células Pequeñas/mortalidad , Carcinoma de Células Pequeñas/patología , Carcinoma de Células Pequeñas/radioterapia , Carcinoma de Células Pequeñas/cirugía , Carmustina/farmacología , Cisplatino/administración & dosificación , Cisplatino/farmacología , Terapia Combinada , Ciclofosfamida/administración & dosificación , Ciclofosfamida/farmacología , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Etopósido/administración & dosificación , Etopósido/farmacología , Estudios de Evaluación como Asunto , Humanos , Lomustina/administración & dosificación , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/cirugía , Mecloretamina/farmacología , Metotrexato/administración & dosificación , Cuidados Paliativos , Valor Predictivo de las Pruebas , Estudios Prospectivos , Manejo de Especímenes , Análisis de Supervivencia , Resultado del Tratamiento , Células Tumorales Cultivadas/efectos de los fármacos , Vincristina/administración & dosificación , Vincristina/farmacología

RESUMEN

Sodium channels of human small-cell lung cancer (SCLC) cells were examined with whole-cell and single-channel patch clamp methods. In the tumor cells from SCLC cell line NCI-H146, the majority of the voltage-gated Na+ channels are only weakly tetrodotoxin (TTX)-sensitive (Kd = 215 nM). With the membrane potential maintained at -60 to -80 mV, these cells produced all-or-nothing action potentials in response to depolarizing current injection (> 20 pA). Similar all-or-nothing spikes were also observed with anodal break excitation. Removal of external Ca2+ did not affect the action potential production, whereas 5 microM TTX or substitution of Na+ with choline abolished it. Action potentials elicited in the Ca(2+)-free condition were reversibly blocked by 4 mM MnCl2 due to the Mn(2+)-induced inhibition of voltage-dependent sodium currents (INa). Therefore, Na+ channels, not Ca2+ channels, underlie the excitability of SCLC cells. Whole-cell INa was maximal with step-depolarizing stimulations to 0 mV, and reversed at +45.2 mV, in accord with the predicted Nernst equilibrium potential for a Na(+)-selective channel. INa evoked by depolarizing test potentials (-60 to +40 mV) exhibited a transient time course and activation/inactivation kinetics typical of neuronal excitable membranes; the plot of the Hodgkin-Huxley parameters, m infinity and h infinity, also revealed biophysical similarity between SCLC and neuronal Na+ channels. The single channel current amplitude, as measured with the inside-out patch configuration, was 1.0 pA at -20 mV with a slope conductance of 12.1 pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (LES), which are known to inhibit ICa and INa in bovine adrenal chromaffin cells, also significantly inhibited INa in SCLC cells. These results indicate that (i) action potentials in human SCLC cells result from the regenerative increase in voltage-gated Na+ channel conductance; (ii) fundamental characteristics of SCLC Na+ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na+ channels are an additional target of the pathological IgG present in the patients' sera.

Asunto(s)

Inmunoglobulina G/farmacología , Bloqueadores de los Canales de Sodio , Carcinoma de Células Pequeñas/fisiopatología , Humanos , Inmunoglobulina G/biosíntesis , Transporte Iónico/efectos de los fármacos , Síndrome Miasténico de Lambert-Eaton/inmunología , Neoplasias Pulmonares/fisiopatología , Potenciales de la Membrana , Canales de Sodio/fisiología , Células Tumorales Cultivadas

RESUMEN

Murine lung epithelial (MLE) cell lines representing the distal bronchiolar and alveolar epithelium were produced from lung tumors generated in transgenic mice harboring the viral oncogene simian virus 40 (SV40) large tumor antigen under transcriptional control of a promoter region from the human surfactant protein C (SP-C) gene. The cell lines exhibited rapid growth, lack of contact inhibition, and an epithelial cell morphology for 30-40 passages in culture. Microvilli, cytoplasmic multivesicular bodies, and multilamellar inclusion bodies (morphologic characteristics of alveolar type II cells) were detected in some of the MLE cell lines by electron microscopic analysis. The MLE cells also maintained functional characteristics of distal respiratory epithelial cells including the expression of surfactant proteins and mRNAs and the ability to secrete phospholipids. Expression of the exogenous SV40 large tumor antigen gene was detected in all of the generated cell lines. The SP-C/SV40 large tumor antigen transgenic mice and the MLE cell lines will be useful for the study of pulmonary surfactant production and regulation as well as lung development and tumorigenesis.

Asunto(s)

Proteolípidos/genética , Surfactantes Pulmonares/genética , Sistema Respiratorio/citología , Animales , Línea Celular , Transformación Celular Viral , Células Epiteliales , Expresión Génica , Pulmón/citología , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Microscopía Electrónica , ARN Mensajero/genética , ARN Viral/genética , Virus 40 de los Simios

RESUMEN

We attempted to prospectively select individualized chemotherapy for 165 non-small cell lung cancer patients based on in vitro analysis of neuroendocrine (NE) markers and drug sensitivity testing (DST) using fresh tumor. The chemotherapy used for small cell lung cancer (SCLC) was selected when NE marker expression determined by L-dopa decarboxylase assay was documented. Selection of chemotherapy for other patients was guided by DST results using a modified dye exclusion assay when available; otherwise etoposide and cisplatin was administered. A total of 112 of 165 (68%) specimens were assayed for L-dopa decarboxylase and 36 patients (22%) had DST. In vitro data directed management for 27 of 96 (28%) patients given chemotherapy: 6 with NE markers were treated with the SCLC regimen; and 21 (58% of those with DST) received their DST-selected chemotherapy regimen. There were no significant differences in response rate among all 3 treatment arms (P = 0.076). However, response to chemotherapy for the patients treated prospectively with a SCLC regimen was 3 of 6 (50%), marginally better than patients given their DST-selected chemotherapy regimen (2 of 21; 9%; P = 0.056) or those treated with etoposide and cisplatin (10 of 69; 14%; P = 0.061). When patients whose NE markers were identified retrospectively are included, 4 of 9 (44%) responded to administered chemotherapy, compared to 7 of 55 (13%) with no NE markers present (P = 0.04). There were no differences in survival among the three treatment groups. Cisplatin and etoposide comprised the most active regimen in vitro for tumors from 16 of 36 (44%) patients, potentially limiting the benefit of DST since this is often the empiric therapy for non-SCLC. Furthermore, the correlation between in vitro and clinical response is nonsignificant for all drugs tested, highlighting the overall relative resistance of non-SCLC tumors to currently available chemotherapy.

Asunto(s)

Antineoplásicos/toxicidad , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/análisis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Sistemas Neurosecretores/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Supervivencia Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Dopa-Decarboxilasa/análisis , Etopósido/administración & dosificación , Femenino , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios Prospectivos , Análisis de Supervivencia , Células Tumorales Cultivadas

RESUMEN

The enzyme DT-diaphorase (DTD; NAD(P)H:quinone oxidoreductase, EC 1.6.99.2), is an obligate two electron reductase which catalyzes reduction of a broad range of substrates, including quinones. We report here variations in DTD concentrations among different classes of lung tumors known also to vary in their responsiveness to cytotoxic agents. Small cell lung carcinomas (SCLCs) and cell lines derived from them have the low DTD activities and mRNA content characteristic of normal human lung, whereas non-small cell lung carcinomas (NSCLCs) have greatly elevated levels. DTD activity was increased up to 80-fold in NSCLC tumors relative to normal lung and 20-35-fold in NSCLC relative to SCLC cell lines. Increased DTD activity appeared to be a function of the NSCLC phenotype rather than a result of derivation from a cell type rich in DTD, since all histological classes of NSCLC showed this phenotype. In addition, where transfection of SCLC cell lines with the v-Ha-ras protooncogene caused a transition to a NSCLC phenotype, DTD activity was also elevated. Neuroendocrine-positive cells (SCLC, carcinoids, and a few NSCLC lines) typically had far lower DTD activities than did cell lines which lacked neuroendocrine markers (most NSCLC cells and mesotheliomas). High DTD activity may be exploited in the design of drugs which undergo bioreductive activation by this enzyme. Consistent with this, xenografts derived from NSCLC cell lines with high DTD that were grown in athymic nude mice were more susceptible to the antitumor quinone, mitomycin C, than were xenografts derived from SCLC cells containing low DTD. These data provide a mechanistic basis for the rational design of more effective bioreductive antitumor agents for use against NSCLC.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/enzimología , Neoplasias Pulmonares/enzimología , Mitomicina/uso terapéutico , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/enzimología , Expresión Génica , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Ratones Desnudos , NAD(P)H Deshidrogenasa (Quinona)/genética , Trasplante de Neoplasias , ARN Mensajero/genética , ARN Neoplásico/genética , Trasplante Heterólogo , Células Tumorales Cultivadas/enzimología

RESUMEN

Utilizing the whole-cell patch-clamp method we assessed the Ca2+ current (ICa) in well-established cell lines from human small-cell carcinoma (SCC) of the lung, NCI-H209 and NCI-H187. The Ca2+ current was readily observed in H209 tumour cells (90% of the cells tested), whereas H187 tumour cells only occasionally expressed Ca2+ channels (26% of the cells tested). H209 Ca2+ current was evoked by potentials greater than -30 mV and exhibited partial inactivation over the duration of a 40 ms command potential. This inward current was unchanged by alteration of the holding potential from -80 to -40 mV and the activation phase of the Ca2+ current was best fitted by Hodgkin-Huxley m(t)2 kinetics. H209 Ca2+ current was reduced over 80% by verapamil (100 microM), whereas w-conotoxin (5 microM) appeared to be without effect. In contrast, H209 Ca2+ current was rapidly abolished by nifedipine (10 microM), strongly suggesting the presence of L-type Ca2+ channels. Voltage-gated Ca2+ channels may be important to the secretion of ectopic hormones and the etiology and pathogenesis of Lambert-Eaton syndrome, an autoimmune disorder of the motor nerve terminal in which autoantibodies directed against voltage-gated Ca2+ channels are produced.

Asunto(s)

Canales de Calcio/metabolismo , Carcinoma de Células Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Canales de Calcio/efectos de los fármacos , Humanos , Síndrome Miasténico de Lambert-Eaton/etiología , Potenciales de la Membrana , Nifedipino/farmacología , Células Tumorales Cultivadas/metabolismo , Verapamilo/farmacología

RESUMEN

We screened 77 non-small-cell lung cancer (NSCLC) cell lines for mutations of the p53 gene using a single-strand conformation polymorphism (SSCP) assay. We found that 57 cell lines (74%) had mutations of the p53 gene. Three cell lines had a deletion of the p53 gene. Of the remaining 54 cell lines, 49 cell lines were sequenced and 52 mutations were confirmed. In contrast to previously published p53 mutations in other human tumors, the p53 gene mutations in NSCLC were diverse with regard to the location and nature of the mutations. The region corresponding to codons 144-166, which is outside the evolutionarily conserved regions, was a frequent site of p53 gene mutations in NSCLC. The presence of a p53 gene mutation was not associated with age, sex, histological types, culture site, treatment intent, presence of prior cytotoxic treatment, neuroendocrine differentiation, median culture time or patient survival. The prevalence of p53 mutations in cell lines with ras mutations did not differ from that in cell lines without ras mutations. However, p53 gene mutations in NSCLC cell lines with ras mutations tended to cluster in exon 8, suggesting the presence of a functional domain of the p53 gene relating to interaction with the ras gene. We conclude that p53 and ras mutations are frequent and apparently independent genetic alterations which play different roles in the pathogenesis, progression and prognosis of NSCLC.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/genética , Genes p53/genética , Genes ras/genética , Neoplasias Pulmonares/genética , Mutación/genética , Secuencia de Aminoácidos , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Exones , Femenino , Pruebas Genéticas , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Células Tumorales Cultivadas

RESUMEN

We analyzed 66 non-small cell lung cancer cell lines for mutations at codons 12, 13, and 61 of all three ras genes and correlated the findings with patient survival. We used designed restriction fragment-length polymorphisms to detect mutations after amplification of ras-specific sequences by the polymerase chain reaction. We found 19 mutations of ras genes (29%), and 11 of these 19 (58%) were at codon 12 of the K-ras gene. By univariate analysis, the presence of any ras mutation in cell lines from patients who received curative intent treatment was associated with a shorter survival (P2 = 0.002). For patients who received only palliative treatment, detection of K-ras mutations at codon 12 was associated with a shortened survival (P2 = 0.0103), but this analysis was not statistically significant for the group with any ras mutation (P2 = 0.093). The Cox proportional hazards model also predicted a higher risk for patients with any type of ras mutations. We conclude that ras mutations, present in a subset of non-small cell lung cancers, are independently associated with the shortened survival of patients, irrespective of treatment intent.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Análisis de Varianza , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Genes ras/genética , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Mutación/genética , Oncogenes/genética , Pronóstico , Modelos de Riesgos Proporcionales , Células Tumorales Cultivadas

RESUMEN

Twenty-five non-small cell lung cancer (NSCLC), 42 small cell lung carcinoma (SCLC), one extrapulmonary small cell carcinoma, 4 carcinoid, and 13 non-lung cancer cell lines were analyzed for human chorionic gonadotropin (HCG) and related glycoprotein hormones. HCG or its subunits were present in 72% of NSCLC, 10% of SCLC, one extrapulmonary small cell carcinoma, 3/4 carcinoids and 2/13 non-lung cancer cell lines. Related glycoprotein hormones were undetectable. These data indicate a frequent production of HCG or its subunits by NSCLC cell lines and cell lines from tumors with carcinoid features. They confirm the clinical inclusion of carcinoids under the broad category of NSCLC rather than SCLC despite their neuroendocrine features. Clinicians should not assume that undifferentiated NSCLC with HCG production represent germ cell tumors.

Asunto(s)

Biomarcadores de Tumor/análisis , Tumor Carcinoide/química , Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Células Pequeñas/química , Gonadotropina Coriónica/análisis , Neoplasias Pulmonares/química , Línea Celular/química , Hormona Folículo Estimulante/análisis , Humanos , Hormona Luteinizante/análisis , Tirotropina/análisis

RESUMEN

Clara cells and type II pneumocytes are the progenitor cells of the bronchioles and alveoli, respectively. These peripheral airway cells (PAC) contain characteristic cytoplasmic structures and express surfactant associated proteins. PAC cell markers are expressed by many pulmonary adenocarcinomas having papillary and/or lepidic growth patterns, which are characteristics of the bronchioloalveolar and papillary subtypes. We investigated the expression of PAC markers in a panel of 41 lung cancer cell lines. Ultrastructural studies demonstrated the presence of cytoplasmic structures characteristic of Clara cells or of type II pneumocytes in 9 of 34 (26%) non-small cell lung cancer cell lines, including 7 of 17 (41%) adenocarcinomas, one squamous cell carcinoma, and one large cell carcinoma. Of interest, the cytoplasmic structures were present in 5 of 6 (83%) cell lines initiated from papillolepidic adenocarcinomas. In addition, we examined the lines for expression of the surfactant associated proteins SP-A, SP-B, and SP-C. Eight of the nine cell lines containing cytoplasmic inclusions characteristic of PAC cells also expressed protein and/or RNA of SP-A, the major surfactant associated protein. Five of these lines expressed SP-B RNA (either constitutively or after dexamethasone induction), while a single line expressed SP-C only after dexamethasone induction. None of six small cell lung cancer cell lines examined expressed any of the PAC markers. Thus, PAC markers are expressed frequently (but not exclusively) in pulmonary adenocarcinoma cell lines, especially in those initiated from tumors having papillolepidic growth patterns. The establishment and identification of multiple cell lines expressing PAC features provide an important new resource for biological and preclinical therapeutic studies.

Asunto(s)

Adenocarcinoma/patología , Carcinoma/patología , Neoplasias Pulmonares/patología , Proteolípidos/análisis , Surfactantes Pulmonares/análisis , Sistema Respiratorio/patología , Células Tumorales Cultivadas/citología , Adulto , Animales , Carcinoma/clasificación , Línea Celular , Sondas de ADN , Dexametasona/farmacología , Glicoproteínas/análisis , Humanos , Neoplasias Pulmonares/ultraestructura , Ratones , Ratones Desnudos , Proteolípidos/genética , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/genética , Sistema Respiratorio/citología , Trasplante Heterólogo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo

RESUMEN

We established a continuous cell line, NCI-H295, from an invasive primary adrenocortical carcinoma. The cell line was established in a fully defined medium (HITES) and later could be adapted for growth in a simple medium supplemented only with selenium, insulin, and transferrin and devoid of serum, steroids, fibroblast growth factor, and a source of exogenous cholesterol. NCI-H295 cells had a relatively long population doubling time and were tumorigenic when inoculated s.c. into athymic nude mice. The cultured cells had ultrastructural features of steroid-secreting cells and contained complex cytogenetic abnormalities including the presence of multiple marker chromosomes. Steroid analyses (radioimmunoassays and mass spectrometry), performed 7 to 9 years after culture initiation, demonstrated secretion of more than 30 steroids characteristic of adrenocortical cells. Total unconjugated steroid secretion in serum-supplemented medium was 2.83 micrograms/10(6) cells/24 h and about 4-fold less in serum-free medium. The major pathway of pregnenolone metabolism in NCI-H295 cells is androgen synthesis, with formation of dehydroepiandrosterone, androstenedione, testotesterone, and at least three sulfated androgens, as well as estrogens. In addition, formation of cortisol, corticosterone, aldosterone, and 11 beta-hydroxyandrostenidione indicated the presence of 11 beta-hydroxylase. Thus, multiple pathways of steroidogenesis are expressed by NCI-H295 cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens. Our findings indicate the presence in NCI-H295 cells of all of the major adrenocortical enzyme systems, including 11 beta-hydroxylase, desmolase, 21 alpha-hydroxylase, 17 alpha-hydroxylase, 18-hydroxylase, lyase, sulfokinase, and aromatase. The NCI-H295 cell line should prove of value in studying the regulation, metabolic pathways, and enzymes involved in steroid formation and secretion. In addition, it may provide insights into the biology and treatment of adrenocortical carcinoma.

Asunto(s)

Neoplasias de la Corteza Suprarrenal/patología , Esteroides/metabolismo , Células Tumorales Cultivadas/citología , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/ultraestructura , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Bandeo Cromosómico , Técnicas de Cultivo/métodos , Humanos , Cariotipificación , Espectrometría de Masas , Ratones , Ratones Desnudos , Microscopía Electrónica , Esteroides/biosíntesis , Trasplante Heterólogo

RESUMEN

Lung carcinoma cell lines were analyzed in culture and in nude mouse xenograft for both morphological appearance and expression of specific proteins that participate in cross-linked envelope formation during normal squamous cell terminal differentiation. Cross-linked envelope formation, induced by artificial influx of millimolar Ca2+ into the cultured cells, was an exclusive trait of squamous, adenosquamous, and mucoepidermoid carcinomas. Small cell lung carcinoma and non-squamous non-small cell lung carcinoma lines, such as adenocarcinoma and large cell carcinoma, were uniformly negative for cross-linked envelope formation. Involucrin, which is incorporated into the cross-linked envelope by the enzyme transglutaminase, was expressed at highest levels in squamous tumors, but several of the non-squamous non-small cell lung carcinoma lines also expressed comparable amounts. On the other hand, transglutaminase activity was consistently higher in squamous as opposed to non-squamous lines, so that in cell culture, a clear contrast between the groups could be observed. A Mr 195,000 protein that is incorporated into cultured human epidermal cell cross-linked envelopes was also observed in some but not all of the squamous lines. Two forms of transglutaminase are expressed in cultured keratinocytes. One of them, tissue transglutaminase, was expressed in the majority of squamous cell lines even though it is not a normal product of squamous differentiation in vivo. Keratinocyte transglutaminase, which is distinct from the tissue form and is normally expressed during terminal differentiation in squamous epithelia. was measurably present in only one of the six squamous cell lines tested. In nude mouse xenografts, keratinocyte transglutaminase, localized immunohistochemically with a biotinylated mouse monoclonal antibody, was again present only in a minority of the squamous lines whereas involucrin was expressed in all. In contrast to involucrin, keratinocyte transglutaminase is not an obligatory component of squamous differentiation in the pulmonary carcinoma cell lines tested. Its expression may be of value in further refining their classification.

Asunto(s)

Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Neoplasias Pulmonares/patología , Precursores de Proteínas/análisis , Transglutaminasas/metabolismo , Animales , Carcinoma de Células Escamosas/análisis , Carcinoma de Células Escamosas/enzimología , Diferenciación Celular , Línea Celular , Citosol/análisis , Humanos , Neoplasias Pulmonares/análisis , Neoplasias Pulmonares/enzimología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante Heterólogo , Células Tumorales Cultivadas/análisis , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/enzimología

RESUMEN

We devised a novel clinical protocol for extensive-stage small cell lung cancer (SCLC), selecting chemotherapy whenever possible on the basis of in vitro drug-sensitivity testing (DST) of individual patients' tumor specimens. Most of the specimens were obtained from metastatic sites during routine staging procedures. Increase of tumor cell number by culture in selective media usually was required before DST could be performed. We used the Weisenthal dye exclusion assay to place the seven drugs in rank order and to select the in vitro best regimen (IVBR), a three-drug combination of proved efficacy in SCLC. After initial staging and specimen acquisition, patients received etoposide and cisplatin (primary therapy) and were restaged after 12 weeks. Patients with partial or no responses and those relapsing after a complete response to primary therapy were switched to the IVBR if DST data were available. If DST data were unavailable, an empiric combination, vincristine-doxorubicin-cyclophosphamide, was administered as secondary therapy. Tumor-containing specimens were collected from 60 of the 80 patients (75%). One or more cell lines were established from 28 patients, and DST data were available from 26 patients (33% of total). Several parameters of in vitro drug sensitivity were significantly associated [two-sided P (P2) less than .05] with clinical response to primary therapy and also with response to the IVBR and were marginally associated with length of survival (.07 less than or equal to P2 less than or equal to .08). Sixteen patients (23%) received their IVBR as secondary therapy, and four of these (25%) attained a complete response, compared with three of 43 (7%) who received an empiric regimen (P2 = .16). We concluded that (a) selection of individualized chemotherapy is labor intensive but feasible in extensive-stage SCLC; (b) DST data are associated with clinical response to primary therapy and to secondary therapy with an IVBR; and (c) further observations will be required if we are to determine whether there is a modest therapeutic benefit to administering the IVBR as a secondary therapy.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Pequeñas/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Pulmonares/tratamiento farmacológico , Carcinoma de Células Pequeñas/mortalidad , Carcinoma de Células Pequeñas/patología , Protocolos Clínicos , Ensayos Clínicos como Asunto , Colorantes , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Estadificación de Neoplasias , Estudios Prospectivos , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

The ability to establish a continuously growing tumor cell line from fresh tumor specimens has been associated with shortened survival in some human malignancies. Therefore, we assessed the relationship between survival and in vitro tumor cell growth from specimens obtained during routine staging procedures in 68 consecutive patients with untreated, extensive-stage small-cell lung cancer (SCLC) who received etoposide/cisplatin chemotherapy. Three groups of SCLC patients could be distinguished: (1) 23 patients in whom a tumor cell line was established in vitro; (2) 28 patients in whom tumor-containing specimens were cultured but in vitro growth did not occur; and (3) 17 patients in whom no tumor-containing specimen could be procured. No significant difference in response rates to chemotherapy of the three groups was noted. Poor performance status (P2 = .001), male gender (P2 = .0008), liver metastases (P2 = .0033), brain metastases (P2 = .0152), and the ability to obtain a tumor-containing specimen from the patient for laboratory culture (P2 = .0005) were all significant independent predictors of decreased survival in this patient population. While the ability to obtain a tumor cell specimen for cell culture using routine staging and diagnostic procedures identified patients with shortened survival, we found no significant survival differences between patients whose tumor cell specimens grew in cell culture v those that did not (median survival of 7 months v 11 months, P2 = .72). Our study indicates that the clinical outcome of extensive-stage SCLC patients from whom tumor cell lines can be established is not significantly different than in those cases from whom tumor-containing specimens could not be grown in vitro.

Asunto(s)

Carcinoma de Células Pequeñas/patología , Neoplasias Pulmonares/patología , Células Tumorales Cultivadas/citología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/mortalidad , División Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Ensayos Clínicos como Asunto , Etopósido/administración & dosificación , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Masculino , Pronóstico , Estudios Prospectivos , Distribución Aleatoria

RESUMEN

We have characterized 14 human colorectal carcinoma cell lines established from primary and metastatic sites by us during the years 1982 to 1985. Five lines were established in fully defined ACL-4 medium and 9 in serum supplemented R10 medium. However, after establishment, cultures could be grown interchangeably in either medium. The lines grew as floating cell aggregates in ACL-4 medium, while most demonstrated substrate adherence in R10 medium. The lines had relatively long doubling times and low cloning efficiencies. Twelve were tumorigenic in athymic nude mice when injected s.c., and two grew i.p. as well. Based on culture, xenograft, and ultrastructural morphologies, the 14 lines could be subtyped as follows: 4 were well differentiated; 5 were moderately differentiated; 4 were poorly differentiated; and 1 was a mucinous carcinoma. Membrane associated antigens characteristic for gastrointestinal cells (carcinoembryonic antigen, CA 19-9, and TAG-72 antigens) were expressed by 50-71% of the lines. Lines expressing carcinoembryonic antigen and CA 19-9 actively secreted these antigens into the supernatant fluids while TAG-72 antigen was not secreted. Surprisingly, 5 of 7 of the original tumor samples tested and 13 of 14 cultured lines expressed L-dopa decarboxylase activity, which is a characteristic enzyme marker of neuroendocrine cells and tumors. In addition, one poorly differentiated cell line contained dense core granules, characteristic of endocrine secretion. Preliminary cytogenetic analyses indicated that 9 of 11 lines examined contained double minute chromosomes. In addition, 3 of the 9 lines with double minutes also had homogeneously staining regions. These findings indicate a high incidence of amplification of one or more as yet unidentified genes.

Asunto(s)

Neoplasias del Colon/patología , Neoplasias del Recto/patología , Antígenos de Neoplasias/análisis , Antígenos de Carbohidratos Asociados a Tumores , Antígeno Carcinoembrionario/análisis , Línea Celular , Cromosomas , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Dopa-Decarboxilasa/análisis , Amplificación de Genes , Glicoproteínas/análisis , Humanos , Neoplasias del Recto/enzimología , Neoplasias del Recto/genética , Células Tumorales Cultivadas

RESUMEN

Human lung cancer cell lines, established from patients with both small cell cancer (SCLC) and non-small cell cancer (non-SCLC; squamous cell, large cell, anaplastic, and adenocarcinoma), were tested for their in vitro chemosensitivity to a panel of drugs. Drug sensitivity was assayed by either soft agar clonogenicity or a novel dye-exclusion assay. Eleven non-SCLC lines (eight continuous, three recently cultured) and five SCLC lines (all continuous) were tested. Four of eight continuous non-SCLC lines cloned sufficiently to permit limited in vitro drug testing, as did two of the five SCLC lines. All 16 cell lines could be tested for multiple drugs using the dye-exclusion assay. Drug concentrations for the nonclonogenic assay more closely approximated the area under the concentration-time curve for a given concentration of each agent. There was considerable variation in the relative sensitivity of the cell lines and the patterns of individual drug sensitivity. The majority of non-SCLC cell lines were refractory to most drugs. Cell lines derived from two previously treated SCLC patients and from three untreated SCLC patients showed greater sensitivity. Concurrent clonogenic and dye-exclusion assays showed similar drug rankings but different absolute values for percent survival. The nonclonogenic dye-exclusion assay is more rapid than the soft agar clonogenic assay (4 days vs. 2-3 weeks), could be performed on all cell lines tested, and appears to reflect the clinical diversity of human lung cancer.

Asunto(s)

Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Pequeñas/patología , Ensayo de Unidades Formadoras de Colonias , Neoplasias Pulmonares/patología , Ensayo de Tumor de Célula Madre , Biotransformación , División Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclofosfamida/farmacología , Evaluación Preclínica de Medicamentos/métodos , Humanos

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Células Cultivadas , Medios de Cultivo , Humanos

RESUMEN

Using a serum-free defined medium, we have established a human cell line, NCI-H929, from a malignant effusion occurring in a patient with IgAk myeloma. The cultured cells have the morphologic, ultrastructural, biochemical, immunologic, and cytochemical features of plasma cells. The cells have rearranged alpha and kappa genes and synthesize and secrete high amounts of IgAk (greater than 80 micrograms/10(6) cells per 24 hours). The cells express surface immunoglobulin (alpha and kappa), the plasma cell antigen PCA-1, the transferrin receptor (T9) and T10 but lack antigens associated with earlier stages of B cell development (HLA-DR, B1, B2, B4, CALLA), as well as other leukocyte-macrophage antigens and Epstein-Barr virus (EBV) nuclear antigen. Although molecular studies confirm that both the tumor and cultured cells are derived from the same clone of malignant B cells, the tumor cells were predominantly near-diploid, whereas the cultured cells are predominantly near-tetraploid with six copies of chromosome 8, four to six of which have an 8q + abnormality. However, both the tumor and the cultured cells have a rearrangement of the cellular c-myc proto-oncogene (located at 8q24) and express c-myc RNA. Although a modest number of human "plasmacytoid" cell lines have been established, most are lymphoblastoid lines lacking plasma cell features, while others appear to be early secretory cells. In contrast, NCI-H929 is a differentiated, highly secretory human plasma cell line.

Asunto(s)

Desoxirribonucleasas de Localización Especificada Tipo II , Mieloma Múltiple/genética , Proto-Oncogenes , Linfocitos B/inmunología , Línea Celular , Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasa HindIII , Epítopos/análisis , Femenino , Regulación de la Expresión Génica , Humanos , Inmunoglobulinas/genética , Cariotipificación , Microscopía Electrónica , Persona de Mediana Edad , Mieloma Múltiple/ultraestructura , Proto-Oncogenes Mas