Host-range properties of murine xenotropic and ecotropic type-C viruses.

Host-range properties of xenotropic (x-tropic) and ecotropic mouse type-C viruses were determined. NB-tropic viruses replicated only in mouse and rat cells, whereas x-tropic viruses, which do not exogenously infect mouse cells, grew in cells from various other mammalian species. Great variability in susceptibilities to x-tropic viruses was demonstrated in cells from heterologous species and in cells from a single species (human). Although rat cells were susceptible to both types of viruses, hamster cells were uniformly resistant. In addition, the x-tropic viruses crossed class barriers to infect cells of avian species but not insect, fish, or reptile cells.

Correlation of in vitro drug-sensitivity testing results with response to chemotherapy and survival in extensive-stage small cell lung cancer: a prospective clinical trial.

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.

Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium.

We tested the ability of serum-free media to support the in vitro growth of human non-small cell lung carcinoma. A medium containing insulin, transferrin, sodium selenite, hydrocortisone, epidermal growth factor, and bovine serum albumin (1 mg/ml) with serum precoating of culture dishes (modified LA medium) supported three previously established cell lines of non-small cell lung cancer and prevented fibroblast proliferation in fresh tumor specimens but did not support long term tumor cell growth from fresh specimens. We added triiodothyronine, sodium pyruvate, and additional glutamine, insulin, and epidermal growth factor to modified LA medium, precoated with fibronectin and collagen instead of serum, and deleted bovine serum albumin, defining a new medium called ACL-3. ACL-3 medium alone supported the short term growth of 10 of 12 cell lines and the soft agarose cloning of 9 of 12 cell lines tested, and ACL-3 supplemented by an optimal concentration of bovine serum albumin (5 mg/ml) supported the long term growth of 10 of 12 cell lines tested. Moreover, we have grown tumor cells for more than 6 months from 11 of 33 (33%) consecutive fresh clinical specimens of human lung adenocarcinoma in ACL-3 with bovine serum albumin. ACL-3 medium provides a defined environment for the study of growth factor requirements of human non-small cell lung cancer and enhances our ability to grow human lung cancer, particularly adenocarcinoma, in vitro.

Cross-linked envelope-related markers for squamous differentiation in human lung cancer cell lines.

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.

Characteristics of cell lines established from human colorectal carcinoma.

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.

Peripheral airway cell differentiation in human lung cancer cell lines.

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.

ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent.

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.

Individualized chemotherapy for patients with non-small cell lung cancer determined by prospective identification of neuroendocrine markers and in vitro drug sensitivity testing.

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.

Elevated DT-diaphorase activity and messenger RNA content in human non-small cell lung carcinoma: relationship to the response of lung tumor xenografts to mitomycin Cl.

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.

Enhanced sensitivity to 1-beta-D-arabinofuranosylcytosine and topoisomerase II inhibitors in tumor cell lines harboring activated ras oncogenes.

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.

Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis.

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.

p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features.

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.

Lack of relationship between in vitro tumor cell growth and prognosis in extensive-stage small-cell lung cancer.

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.

Cytosolic insulin-degrading activity in islet-derived tumor cell lines and in normal rat islets.

RIN-m cells, cultured from a rat insulinoma, not only bind and secrete but also degrade insulin (Diabetes 1982; 31:521-31). The insulin-degrading activity resides in the cytosol and is similar to the insulin-specific proteases previously described in muscle and other tissues. It has an apparent Km of 0.15 microM for porcine insulin in crude cell-free extracts, a competitive inhibition constant for proinsulin that is close to the Km, and a lower but measurable affinity for glucagon. The enzyme is inactive at pHs below 6.0, indicating that it is not lysosomal, is completely inhibited by N-ethylmaleimide, and exhibits apparent competitive inhibition constants (microM) for the following peptides: desoctapeptide insulin, 0.043; guinea pig insulin, 0.048; proinsulin, 0.64; insulin B-chain, 1.17; glucagon, 7.0; and cyclic somatostatin, 8.6. Highly active insulin-degrading activity was found using cell suspensions of 22 cloned and 8 subcloned cell lines derived from RIN-m as well as 11 other continuous cell lines derived from a variety of nonislet tissues of rat, mouse, and human origin. Homogenates of the original rat islet tumor and cytosol of normal rat islets also contained insulin-degrading activity. Although insulin protease is present in a variety of tissues, it may have an additional regulatory function in cells that are actively synthesizing, storing, and secreting insulin.

Insulin, glucagon, and somatostatin receptors on cultured cells and clones from rat islet cell tumor.

Cells grown in culture from rat islet cell tumor (parent cells) and clones obtained from them were used in this study. Parent cells secreted primarily insulin and somatostatin with very small quantities of glucagon. The clones, based on hormone content and secretion, were divided into three phenotypic groups: insulin secreting, somatostatin secreting, and nonsecreting clones. Specific receptors for insulin, glucagon, and somatostatin were demonstrated on parent cells and clones. Parent cells bound 4.12 +/- 0.46% insulin, 2.11 +/- 0.29% glucagon, and 2.49 +/- 1.24% somatostatin per 2 x 10(6) cells. Characteristic hormone binding patterns were observed in insulin secreting versus somatostatin secreting clones. Insulin secreting versus somatostatin secreting clones. Insulin secreting clones bound less insulin than somatostatin and other noninsulin-secreting clones (P less than 0.02). In contrast, somatostatin secreting clones bound more somatostatin than non-somatostatin-secreting clones (P less than 0.05). Somatostatin-secreting clones had a significantly greater number of receptors for all three hormones. The difficulties involved in the interpretation of the quantitative aspects of binding in the presence of continued hormone secretion are discussed. Nonetheless, the presence of receptors on the cells for hormones secreted by the same cells strongly suggests autoregulation. The apparent low affinity of some of these receptors and the presence of receptors for all three islet cell hormones on all islet cells supports the likelihood of paracrine controls.

Characteristics of the interaction of the glucagon receptor, cAMP, and insulin secretion in parent cells and clone 5F of a cultured rat insulinoma.

Rat insulinoma cells, which grow in culture and secrete insulin, were used to study the mechanism of stimulation of insulin release by glucagon. The parent cell line (RIN-m) and a clone that secretes high levels of insulin (5F) had been shown to possess specific receptors for glucagon. Glucagon (1 microM) stimulated a rapid increase in cyclic adenosine 3':5'-monophosphate (cAMP) that was followed by an increase in insulin secretion in both cell lines. The concentration of glucagon necessary for half-maximal stimulation of cAMP was 50 nM in parent and approximately 0.5 microM in 5F, whereas the concentration required to inhibit binding by 50% was 0.5 nM and 30 nM, respectively. In 5F, the dose-response relationships for cAMP and insulin secretion were superimposable. The glucagon effects on insulin secretion and cAMP did not require either glucose or amino acids in the incubation media. No refractoriness to glucagon stimulation of cAMP or insulin was noted. It may be concluded that there are significant differences between glucagon binding and glucagon responses in parent cells and clone 5F, there are glucagon receptors that are not coupled to adenylate cyclase, and cAMP mediates glucagon-stimulated insulin release.

Clonal analysis of insulin and somatostatin secretion and L-dopa decarboxylase expression by a rat islet cell tumor.

A cell line, RIN-m, established from a transplantable rat islet cell tumor secretes insulin (IRI) and somatostatin (SRIF) and expresses high levels of the key amine precursor uptake and decarboxylation (APUD) cell enzyme L-dopa-decarboxylase (DDC). Conditioned medium from a rat pituitary tumor line GH3, secreting GH and PRL, improved the cloning efficiency of RIN-m cells 24-fold and enabled the isolation and establishment of a large number of primary and secondary clones. These clones were used to study clonal relationships between peptide hormone secretion and APUD features of an endocrine cell. All the primary and secondary clonal derivatives, irrespective of whether they secreted peptide hormones, maintained high levels of DDC activity. In contrast, IRI and SRIF secretion patterns of the primary clones were highly variable. Selective recloning of primary clones resulted in the isolation of subclones which produced either no hormones or high levels of either IRI or SRIF, but no clone that continuously secreted high levels of both IRI and SRIF. We conclude that: 1) the rat pituitary tumor line GH3 produces a factor(s), possibly GH and/or PRL, which dramatically affects the growth and cloning efficiency of rat islet tumor cells; 2) in contrast to the variability in hormone secretion patterns, DDC activity was consistently expressed in all clones and subclones; and 3) although wide fluctuation in hormone secretion levels occurred among the primary clones, subclones were obtained which revealed that IRI and SRIF can be expressed independently. The subclones of RIN-m developed should be useful for the analyses of factors influencing the synthesis, storage, and secretion of IRI and SRIF. The persistence of high DDC activity in the primary and secondary clones suggests that the APUD property of this endocrine cell may be a primitive differentiation feature closely related to the stem cell; in contrast, peptide hormone production may be associated with more terminal differentiation events.

Transfer plate radioassay using adsorbed anti-insulin antibody to detect insulin secreted by islet cell cultures.

A solid-phase radioimmunoassay for detection of insulin synthesized by islet cell clones is described. This assay employs anti-insulin antibody adsorbed onto fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the bottom of each transfer plate well permits fluid to enter the wells when transfer plates are lowered into microculture wells containing insulin. With this assay it is possible to rapidly screen hundreds of islet cell cultures for insulin production. We have used this assay to facilitate cloning of the RIN rat insulinoma cell line. The assay readily detects insulin synthesis by RIN cells and [125I]insulin is not displayed by culture medium from cells which do not produce insulin. The transfer plate format should be applicable to semiautomate other radioimmunoassays.

Human chorionic gonadotropin and related glycoprotein hormones in lung cancer cell lines.

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.

Cell culture methods for human lung cancer.

With the use of defined or partially defined media, most human lung cancers can be successfully cultured. The cultures retain the morphologic, biochemical, and aneuploid properties of the tumors from which they were derived. Thus, short-term or established cell lines are valuable models for indepth study of the biology of human lung cancer.