High levels of intracellular bombesin characterize human small-cell lung carcinoma.

"Small cells" or "oat cells" characterize a virulent form of lung cancer and share many biochemical properties with peptide-secreting neurones. The neuropeptide bombesin is present in all small-cell lines examined, but not in other lung cancer cell lines, suggesting that bombesinergic precursor cells in lung may give rise to this disease.

Transferrin synthesis by small cell lung cancer cells acts as an autocrine regulator of cellular proliferation.

Since transferrin is required for cellular proliferation, we investigated transferrin synthesis by a small cell lung cancer line (NCI-H510) that survives in serum-free media without added transferrin. Immunoassays for human transferrin demonstrated that these cells contained immunoreactive human transferrin. Immunofluorescence studies showed that the protein is expressed on the surface of cells, presumably bound to transferrin receptor. Media conditioned by NCI-H510 cells support proliferation of human leukemic cells that would not survive in media lacking transferrin. [35S]Methionine incorporation documented transferrin synthesis by NCI-H510 cells as well as three other small cell lines. Transferrin synthesis by NCI-H510 cells increased more than 10-fold when cells entered active phases of the cell cycle, and this increase was seen before large increases in transferrin-receptor expression. Further experiments examining the effects of agents that affect iron metabolism show that the addition of transferrin-iron or hemin to the media is associated with a more rapid initial rate of proliferation and lower rates of transferrin synthesis than control cells. Gallium salts, which inhibit iron uptake, inhibited proliferation of these cells. If the cells recovered from this effect, transferrin synthesis remained greatly increased compared to control. We conclude that transferrin synthesis by these malignant cells is ultimately related to an iron requirement for cellular proliferation. It appears that this synthesized transferrin acts as part of an important autocrine mechanism permitting proliferation of these cells, and perhaps permitting tumor cell growth in vivo in areas not well vascularized.

Lectin-binding sites in preneoplastic and neoplastic lesions of human and rodent respiratory tracts.

The localization of fluorescein-labeled lectins, i.e., concanavalin A (Con A), Ricinus communis-120 (RCA), and wheat-germ agglutinin (WGA), were studied histologically in F344 rat epithelial lesions produced in the course of chemical carcinogenesis. WGA could not be demonstrated in these lesions. Although all lesions showed positive-binding sites when high concentrations of either Con A or RCA were used, a dilution study showed that the epithelial lesions had different affinities for lectins. With both Con A and RCA, dysplastic and neoplastic lesions showed the strongest intensity of fluorescence and squamous metaplasia showed the weakest. Normal and hyperplastic epithelia showed intermediate intensity. In the dilution study, RCA showed eight times more affinity and Con A showed two times more affinity for dysplastic and neoplastic epithelia than for normal or hyperplastic epithelium. Similar affinity patterns were observed in human lesions and tumors. With Con A, 58% of tumors showed much stronger fluorescence than did normal epithelium, and 44% of the tumors showed positive fluorescence with RCA. Although both lectins exhibited a stronger affinity for all the dysplastic-neoplastic lesions than for normal or hyperplastic epithelium, RCA proved to be the most adequate marker for preneoplastic lesions.

Retention of chromosome 3 in extrapulmonary small cell cancer shown by molecular and cytogenetic studies.

In small cell lung carcinoma, one of the short arms of chromosome 3 is typically lost. To investigate chromosome 3 in extrapulmonary small cell carcinoma, we used DNA probes that detect restriction-fragment-length polymorphisms at loci on 3p. These probes were used to study DNA extracted from tumors and normal tissues and/or tumor cell lines from five patients with extrapulmonary small cell cancer. Tumor DNA from four of the five patients with extrapulmonary small cell cancer retained heterozygosity at loci on 3p. Cytogenetic studies of the tumor cell lines established from these four patients showed retention of both short arms of chromosome 3. We conclude that the loss of genetic material from 3p observed in small cell lung cancer is not typical in extrapulmonary small cell cancer.

A novel monoclonal antibody, SCCL 175, with specificity for small cell neuroendocrine carcinoma of the lung.

The murine monoclonal antibody SCCL 175, which is one of several monoclonal antibodies directed against small cell neuroendocrine carcinoma developed by one of us (E.D.B.), was studied for its immunohistochemical reactivity against normal human tissues and a spectrum of bronchopulmonary and metastatic carcinomas using the avidin-biotin complex technique. SCCL 175 reacted with 40 of 44 small cell carcinomas including both primary and metastatic sites and was distributed both on the cell surface and intracytoplasmically. Staining was seen in fresh frozen tissues, cytology preparations, and in a limited number of paraffin-embedded tissue sections after trypsin pretreatment. It was nonreactive with all non-small cell lung carcinomas, neuroendocrine carcinomas from other primary sites, and nonpulmonary carcinomata studied to date. Its distribution in normal adult human tissues was limited to some hypothalamic neurons and the apical membranes of renal proximal tubular epithelium. Cytotrophoblastic and syncytotrophoblastic cells from placental tissue demonstrated variable SCCL 175 immunoreactivity. Of choriocarcinomas studied, one of three demonstrated focal staining. These findings demonstrate the diagnostic utility of SCCL 175 in phenotyping small cell carcinoma of lung, and its specificity suggests a potential role in the therapy of this disease.

Keratin subtypes of small cell lung cancer.

The molecular forms of keratin in small cell lung cancer (SCLC) cell lines and tumors were examined with antikeratin monoclonal antibodies. Immunostaining of SCLC by antikeratin antibody and examination by fluorescence microscopy indicates population heterogeneity in keratin content. Intensity of immunostaining is often weak. However, polyacrylamide gel electrophoresis and immunoblotting reproducibly demonstrate the presence of keratin and allow analysis of the keratin subtypes. The finding of keratin subtypes closely associated with the development of keratinizing epithelium (the 68 kDa basic keratin) in SCLC was unexpected in a tumor that is regarded as poorly differentiated. The cytoskeletal composition of SCLC suggests the presence of a heterogeneous population with a significant proportion of cells expressing highly differentiated epithelial properties.

Differential expression of the gene encoding the novel pituitary polypeptide 7B2 in human lung cancer cells.

The protein designated 7B2 is a recently discovered pituitary polypeptide which is selectively expressed in cells containing secretory granules, such as neurons and endocrine cells. Northern blot analysis of 7B2 gene expression in small cell lung carcinoma (SCLC) cell lines revealed that 7B2 was expressed in all nine cell lines of the classic type tested, but in six of seven SCLC cell lines of the variant type, 7B2 expression could not be detected. In only one of four non-SCLC cell lines tested, 7B2 was expressed. Furthermore, in 16 primary human non-SCLCs, no or only very low expression of 7B2 was found. In the eight primary human SCLCs tested, expression of 7B2 appeared variable: three exhibited a high level of expression; three a low level; while in two cases, expression was very low or not detectable at all. Finally, the three carcinoid tumors tested expressed very high levels of 7B2 mRNA. These data indicate that the 7B2 gene is a useful marker not only to discriminate between classic and variant types of SCLC cell lines, but also in human lung cancer diagnosis.

Immunoreactive gastrin-releasing peptide as a specific tumor marker in patients with small cell lung carcinoma.

Gastrin-releasing peptide (GRP) is now known to be a very common product of small cell lung carcinoma (SCLC). With the aim of investigating the possible role of this peptide as a tumor marker of SCLC, we have developed a sensitive radioimmunoassay system for plasma immunoreactive GRP using immune-affinity chromatography for plasma extraction. Plasma immunoreactive GRP levels in control subjects were determined by using 15 ml of plasma as the starting material (minimum concentration detectable, 0.8 pg/ml). The levels in 10 control subjects were (mean +/- SD) 1.2 +/- 0.27 pg/ml; range, 0.86-1.7 pg/ml. This assay system was applied for the clinical use by using 3 ml of plasma as the starting material (minimum concentration detectable, 4.0 pg/ml). Plasma immunoreactive GRP levels were elevated in SCLC patients at frequencies of 71% in patients with limited disease and 80% in those with extensive disease. Furthermore, a change in the level showed excellent correlation with the therapeutic response. In six patients with complete response who had had elevated levels before treatment, the levels decreased to an undetectable range when the tumor disappeared, and they remained undetectable until 1 month later, when the patients were judged to have achieved complete response. In the partial response group, plasma immunoreactive GRP levels had decreased to an undetectable level in two of three patients, when the patients achieved partial response. In four patients with progressive disease, plasma immunoreactive GRP levels were elevated at the time of the progressive disease judgment, when compared with levels before treatment. The levels in 21 patients with non-SCLC (10 with adenocarcinoma, seven with squamous cell carcinoma and four with large cell carcinoma) were not elevated. These results indicate the plasma immunoreactive GRP level as a useful tumor marker in SCLC patients. It is now believed that GRP can function as an autocrine growth factor for SCLC. The present study suggests that the possible autocrine growth factor could serve as a reliable tumor marker for cancer patients.

Occurrence of fibrin and tissue factor antigen in human small cell carcinoma of the lung.

Fibrin was detected by specific immunofluorescence in tissue obtained from five of six cases of small cell carcinoma of the lung. Dense specific fluorescence was observed in the connective tissue stroma surrounding metastatic tumor nodules and frequently in the scant extracellular stroma surrounding individual viable tumor cells and small tumor cell clusters. When observed by electron microscopy, the fibrin hugged tumor cell plasma membranes and, in some areas, seemed to envelop the cells. Fluorescent staining of tumor cells, but not the stroma, was observed with an antibody to tissue factor. These findings suggest that local activation of coagulation occurs in small cell carcinoma of the lung. Deposited fibrin may contribute to the growth and spread of this particular type of cancer.

Epidermal growth factor receptor expression in human lung cancer cell lines.

Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines were studied for epidermal growth factor (EGF) receptor expression. All NSCLC cell lines tested (eight of eight) had specific EGF binding sites, whereas only five of 11 SCLC cell lines bound EGF. NSCLC and SCLC cell lines expressed the same type of high affinity EGF binding sites with a Kd of 0.5 to 4.5 nM; however, NSCLC cells bound significantly more EGF than SCLC cell lines. The amount of binding sites in NSCLC cells ranged between 71 and 1,000 fmol/mg of protein and in SCLC cells, between 26 and 143 fmol/mg of protein. The two SCLC cell lines with EGF binding values within the range of NSCLC belonged to the variant subtype of SCLC. By means of an anti-erbB serum and indirect radioimmunoprecipitation, a strong Mr approximately 170,000 protein band could be detected in the NSCLC cell lines. This protein corresponds to the EGF receptor molecule. Its identity was proven by competition with excess erbB antigen for the antibody during the radioimmunoprecipitation. Furthermore, this Mr 170,000 protein exhibited protein kinase activity as evidenced by in vitro autophosphorylation. The radioactivity incorporated into the Mr 170,000 band in radioimmunoprecipitation and protein kinase assays was 10 to 100 times lower in these SCLC cell lines which were positive in the EGF binding assay compared to the NSCLC cell lines. We conclude that NSCLC in contrast to SCLC expresses high levels of EGF receptors which may be used to facilitate the differential diagnosis in some cases of lung cancer. These data suggest that EGF may play a role in growth and differentiation of NSCLC.

Tumor-associated membrane sialoglycoprotein on human small cell lung carcinoma identified by the IgG2a monoclonal antibody SWA20.

A mouse IgG2a monoclonal antibody, SWA20, defining a tumor-associated cell surface antigen on small cell carcinoma of the lung (SCC) was generated. The reactivity of the antibody with cell lines was examined by indirect immunofluorescence staining and solid phase radioimmunoassay and the reactivity with tissues by immunoperoxidase staining. The antibody reacts with a proportion of small cell carcinoma cell lines (4 of 8) and tissues (7 of 12), but not with other pulmonary or extrapulmonary cell lines (0 of 30) or tumor tissues (0 of 78). The antibody was unreactive with primary cultures of normal bronchial epithelial cells, RBC, and WBC. Immunoperoxidase staining of normal tissues showed rare antigen-positive cells in suprabasal layers of bronchial epithelium and less than 10% of positive cells in colon epithelium. Immunoblots of SCC extracts demonstrated antibody reactivity with a doublet band at Mr 40,000, a broader band at Mr 100,000, and a band at Mr 180,000. The antigen was not present in crude lipid extracts of SCC cells. Solid phase radioimmunoassays and immunoblots showed binding competition with the lectin Triticum vulgaris, sensitivity of the antigen to neuraminidase, and a partial sensitivity to treatment with periodate. The antigen was coexpressed on SCC cell lines with the antigen sGP90-135 defined first by antibody LAM8 (R. Waibel, C. J. O'Hara, and R. A. Stahel. Cancer Res., 47:3766-3770, 1987) but differed from it by lack of reactivity with Lea-positive saliva and partial resistance to periodate treatment. There was no binding competition between radiolabeled antibodies SWA20 and LAM8 to SCC target cells. The IgG2a antibody SWA20 identifies a previously undescribed tumor-associated surface membrane antigen, sGP100, expressed selectively on a proportion of SCC.

Expression of an estramustine-binding associated protein in human lung cancer cell lines.

Estramustine-binding protein has previously been demonstrated in normal rat prostatic tissue, in normal human prostate epithelium, and in prostatic carcinomas. It binds specifically estramustine and estromustine, the cytotoxic metabolites of estramustine phosphate (Estracyt), a drug which is used in the treatment of prostatic carcinoma. In this study we have examined the presence of an estramustine-binding associated protein in a panel of human cell lines, representing the major histopathological types of lung cancer. A mouse (murine) monoclonal antiserum developed against rat estramustine-binding protein was used for immunohistochemical detection. Fast protein liquid chromatography was used for biochemical characterization. As judged from the immunohistochemical investigation, estramustine-binding protein was present in large amounts in five of six non-small cell carcinoma cell lines, while seven of eight small cell carcinoma cell lines were essentially negative. Fast protein liquid chromatography analyses of lysated cells from the lung cancer cell lines, incubated with [3H]estromustine, concurred with the results from the immunohistochemical stainings. These data strongly indicate a convincing connection between the immunoreactivity and ligand-binding properties of estramustine-binding protein in the cell lines examined. The presence of an estramustine-binding associated protein in human lung cancer cell lines has implications for further investigations into the biological relevance and the potential for eventual therapeutic applications.

Identification of tissue factor in two human pancreatic cancer cell lines.

We have studied the effects of two human pancreatic cancer and two human small cell lung cancer cell lines on clotting and platelet aggregation. Both pancreatic lines markedly shortened recalcification times and induced platelet aggregation. The lung cancer lines produced little shortening of recalcification times and no platelet aggregation. The clotting and aggregation activities of the pancreatic lines were further characterized. Recalcification times following the addition of cancer cell line material to plasmas deficient in factors VII and X were markedly prolonged, suggesting that the activity is due to tissue factor. Hirudin, an inhibitor of thrombin from the saliva of leeches, and rabbit polyclonal immunoglobulin G anti-bovine brain tissue factor inhibited both procoagulant and aggregation activities. Apyrase (an enzyme degrading ADP), diisopropylfluorophosphate (a serine protease inhibitor) and L-trans-epoxysuccinylleucylamido(4-guanidino)butane (a cysteine protease inhibitor) failed to inhibit these activities. Increasing concentrations of heparin inhibited platelet aggregation. Subcellular fractionation studies showed these activities to be localized to the plasma membrane. The association between mucin and the acceleration of clotting has been well described. The absence of mucin in electron micrographs of these pancreatic whole cells, membrane fractions, and shed microvesicles, as well as the failure of chaotropic agents (i.e., agents stripping material extrinsic to the cell membrane such as mucin) to abrogate this activity support these activities being intrinsic to the plasma membrane. These data strongly suggest that these activities are due to tissue factor which appears to be released as microvesicles in vitro. The release of tissue factor via microvesicles in vivo is one possible mechanism for the coagulopathy sometimes seen in patients with pancreatic carcinoma.

Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological, and growth properties.

We have described the establishment and biochemical characterization of 50 small cell lung carcinoma (SCLC) cell lines. Further analysis of these data, combined with studies of morphology and growth characteristics, indicates that 35 (70%) of the lines retained typical morphology (SCLC, intermediate subtype), growth characteristics (growth as tightly packed floating cellular aggregates, long doubling times and low colony-forming efficiencies), and biochemical profile (presence of L-dopa decarboxylase, bombesin-like immunoreactivity, neuron-specific enolase, and high concentrations of brain isoenzyme of creatine kinase). They are referred to as classic SCLC lines. The remaining 15 (30%) lines had discordant expression of the biochemical markers; they retained high concentrations of brain isozyme of creatine kinase, but had significantly lower concentrations of neuron-specific enolase and lacked L-dopa decarboxylase and bombesin-like immunoreactivity. These cell lines are called variants. SCLC variant lines could further be divided into (a) biochemical variant lines having variant biochemical profile but retaining typical SCLC morphology and growth characteristics; and (b) morphological variant (SCLC-MV) lines having variant biochemical profile, altered morphology (features of large cell undifferentiated carcinoma) and altered growth characteristics (growth as loosely attached floating aggregates, relatively short doubling times and cloning efficiencies). Fifty-five clones derived from the three SCLC subclasses retained their parental phenotypes. In SCLC-MV lines there was a near constant relationship between variant morphology, altered growth characteristics and amplification of the c-myc oncogene; classic SCLC and biochemical variant SCLC lines were not amplified. Variant morphologies frequently are present in SCLC tumors at autopsy, and most SCLC-MV lines reflect changes that had occurred in the tumors from which they were derived. Because SCLC-MV tumors behave more virulently in the patient and are radioresistant in vitro, these findings are of considerable biological and clinical interest.

A comparison of synaptophysin, chromogranin, and L-dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines.

Synaptophysin is a Mr 38,000 integral membrane glycoprotein expressed by a variety of normal and neoplastic neuroendocrine cells. We studied synaptophysin as an immunocytochemical marker for neuroendocrine differentiation in lung cancer and compared it to the immunocytochemical expression of chromogranin A, a marker for dense core (endocrine) granules, and the biochemical activity of L-dopa decarboxylase (DDC), the key amine-handling enzyme. Of the 250 cell lines available to us, we selected examples representative of the following cell types: bronchial carcinoids (n = 4), small cell lung cancer (SCLC) (n = 7), extrapulmonary small cell carcinomas (n = 4), and non-small cell lung cancers (n = 18) whose neuroendocrine status had been previously determined on the basis of electron microscopy and DDC activity. We demonstrated (a) there was a higher incidence of synaptophysin than chromogranin A immunoreactivity in carcinoid (100 versus 75%), classic SCLC (70 versus 50%), and variant SCLC (57 versus 29%) cell lines; (b) 3 of the 4 (75%) extrapulmonary small cell lung cancer cell lines expressed synaptophysin and chromogranin A; (c) 5 of the 7 (71%) non-small cell lung cancer cell lines previously shown to express multiple neuroendocrine markers were positive for synaptophysin, chromogranin A, and DDC activity; (d) none of the other 11 non-small cell lung cancer cell lines expressed synaptophysin or chromogranin A; and (e) formalin fixation and paraffin embedding reduced synaptophysin immunoreactivity in 11 of 14 (79%) of the cell lines, as compared to freshly prepared specimens fixed in 95% ethanol. Western blot analysis using the synaptophysin antibody (SY38) demonstrated immunoreactive proteins ranging from Mr 43,000 to 45,000 in five representative cell lines. The concordance of expression of all three neuroendocrine markers was statistically significant when values for all cell lines were totalled. Synaptophysin was a more commonly expressed marker for variant SCLC cell lines, which rarely showed DDC activity. We conclude that synaptophysin may be a more sensitive and specific marker for neuroendocrine differentiation, when compared to chromogranin A and DDC in lung cancer cell lines which express only part of the neuroendocrine program.

Production of the alpha subunit of guanine nucleotide-binding protein GO by neuroendocrine tumors.

We have found that neuroendocrine tumors (including neuroblastoma, ganglioneuroma, gut carcinoid, pheochromocytoma, medullary thyroid carcinoma, insulinoma, glucagonoma, prolactinoma, carotid body tumor, and small cell lung carcinoma) produce considerable amounts (about 1000-80,000 ng/g tissue) of the alpha subunit of guanine nucleotide-binding protein, GO (GO alpha), whereas nonneuroendocrine tumors contain less than 300 ng of GO alpha/g tissue. GO alpha in the neuroendocrine tumors was present both in the soluble fraction, and cholate-extractable membrane-bound fraction of tissues. Immunoblots of membrane fractions of neuroblastoma and carcinoid tissues confirmed that the immunoreactive substance in the tumor tissues was GO alpha. Immunohistochemically, GO alpha was localized consistently in the cell membrane and occasionally in the cytoplasm of neuroendocrine tumors. GO alpha was also detected in sera of 73% patients with neuroblastoma at diagnosis, whereas serum GO alpha concentrations in control children, or patients with nonneuroendocrine tumors were lower than the detection limit of the immunoassay method employed. Serum GO alpha concentrations in patients with neuroblastoma changed with the clinical course; they fell in patients responding to treatment and increased in patients who relapsed. Since GO alpha, a specific protein in the neural and neuroendocrine cells, was found to be produced in considerable amounts by all types of neuroendocrine tumors but not in nonneuroendocrine tumors, GO alpha might be a useful biomarker for neuroendocrine tumors.

Voltage-operated calcium channels in small cell lung carcinoma cell lines: pharmacological, functional, and immunological properties.

Different subtypes of voltage-operated calcium channels (VOCCs) are expressed in different tissues and can be distinguished by functional and pharmacological criteria. One type of high voltage-activated calcium channel, specifically recognized by the peptide neurotoxin omega-conotoxin (omega CTx), is expressed only in neurons. Seven different human small cell lung carcinoma (SCC) cell lines were also found to bind 125I-omega CTx. The binding was specific, saturable, and of high affinity. 125I-omega CTx binding was not antagonized by the calcium channel ligands verapamil, nitrendipine, and diltiazem. There was a correlation between the amount of toxin binding and the detection of depolarization-induced calcium fluxes studied with the fluorimetric probe Fura2. Fura2 experiments also demonstrated that, in addition to omega CTx-sensitive calcium channels, SCC cell lines also expressed omega CTx-insensitive calcium channels, which were antagonized by nitrendipine and verapamil. 125I-omega CTx-labeled VOCCs from SCC cells were, furthermore, precipitated by anti-VOCC autoantibodies obtained from patients affected by the Lambert-Eaton myasthenic syndrome, a neuromuscular disease often associated with SCC. The present findings further indicate the presence of neuronal molecules with important biological function on SCC plasma membrane and add new insights into the pathogenetic mechanism of autoimmune neurological paraneoplastic diseases, like Lambert-Eaton myasthenic syndrome.

Characterization of a human small cell lung carcinoma cell line with acquired resistance to cis-diamminedichloroplatinum(II) in vitro.

A 6.4-fold cis-diamminedichloroplatinum(II) (CDDP) resistant human small cell lung carcinoma cell line (GLC4-CDDP) was developed to study acquired CDDP resistance in vitro. Compared to the sensitive cell line (GLC4), the GLC4-CDDP showed an increase in doubling time and a decrease in cloning efficiency, cellular size, double minutes per cell, cellular protein, and nuclear protein content. While a complete cross-resistance for tetraplatin and a partial cross-resistance for doxorubicin, melphalan, cadmium chloride, carboplatin, and cis-dichloro-trans-dihydroxo-cis-bis(isoprolylamine)platinum (IV) (resistance factor, respectively,4.0,5.8,2.1,1.5,2.9) was found, no cross-resistance for vincristine was found. In the GLC4-CDDP line in comparison to the GLC4 line, glutathione and total amount of sulfhydryl compounds was significantly increased, while glutathione S-transferase and glutathione reductase was the same. The platinum content in cells and nuclei was lower in the resistant line, but after correction for cellular protein or volume no difference was found. The amount of platinum bound to DNA was significantly lower in the GLC4-CDDP line. After a 1-h incubation with CDDP, the amount of Pt-GG adducts was the same and the amount of interstrand cross-links was reduced in the GLC4-CDDP line as compared to GLC4. In conclusion, in the GLC4-CDDP line the phenotype and genotype are changed and various mechanisms, such as decreased Pt-DNA binding, elevated glutathione, and reduced interstrand cross-links, play a role in the development of the CDDP resistance.

DNA breakage in human lung carcinoma cells and nuclei that are naturally sensitive or resistant to etoposide and teniposide.

Evidence suggests that the anticancer agents etoposide (VP16-213) and teniposide (VM26) produce DNA breaks and cytotoxicity by interaction with type II topoisomerase. Therefore, levels of type II topoisomerase may influence sensitivity to VP16-213 and VM26. We have characterized four lung carcinoma-derived cell lines for natural sensitivity or resistance to VP16-213 and VM26. Included in this study were two small cell lung carcinoma lines (SW900 and SW1271), an adenocarcinoma line (A549), and a large cell carcinoma (H157). SW1271 was the most sensitive line with a median inhibitory concentration for cell proliferation of 0.5 microM for VM26 and 2.7 microM for VP16-213, and SW900 was the most resistant with median inhibitory concentration values of 2.0 and 16 microM, respectively. A549 and H157 cells were intermediate in sensitivity to these drugs. Alkaline elution techniques were used to study in vivo formation and repair of single and double strand DNA breaks. Single strand DNA breaks were observed in SW1271 cells exposed to as little as 10 nM VM26 or 100 nM VP16-213 for 1 h, whereas SW900 cells required exposure to 10-fold higher concentrations of VM26 or VP16-213 to produce similar results. Single strand DNA breaks predominated only in SW1271 and A549 cells and then, only at low drug concentrations, whereas the ratios between single and double strand DNA breaks decreased at higher drug concentrations. Plots of cytotoxicity versus single and double strand DNA breakage revealed that cytotoxicity produced by both drugs was more closely related to double strand DNA break formation in all four cell lines. DNA breaks appeared rapidly upon addition of drug, reaching plateaus in DNA breaks within 30 min, and repair of both single and double strand DNA breaks occurred rapidly with time to repair one-half of the DNA breaks of 20 to 60 min in all four cell lines upon removal of drug, arguing against repair as a mechanism for drug resistance. DNA breakage was also observed in nuclei isolated from SW900 and SW1271 cells in similar magnitude to that observed in the respective cells. Results indicate that DNA breakage plateaus may reflect a steady-state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and suggest that natural resistance to VP16-213 and VM26 may be due to different enzyme levels in sensitive and naturally resistant cells.

Expression of p64c-myc and neuroendocrine properties define three subclasses of small cell lung cancer.

Twelve human small cell lung cancer (SCLC) cell lines and 6 non-SCLC cell lines were analysed with respect to expression of the c-myc, c-myb, and c-raf1 protooncogenes at the protein level. Analysis of p64c-myc protein expression in 12 SCLC cell lines resulted in the observation that it is present at high levels not only in cells with low, but also in those with moderate neuroendocrine differentiation. Neuroendocrine differentiation was based on parameters such as growth rate, colony formation, L-Dopa decarboxylase (DDC) activity, bombesin, and neurotensin described before. Surprisingly, in two cell lines with low neuroendocrine differentiation but without c-myc protein expression (SCLC-86M1 and NCI-H526) p75c-myb expression was observed which may therefore be able to substitute for the p64c-myc protein. Analysis of p74c-raf1 expression did not result in correlation with any growth or differentiation parameter since it was expressed at low levels in 11 out of 12 cases. We conclude that SCLC in vitro can be classified in three rather than two previously defined subclasses. In addition to the classic subclass with slow growth, high neuroendocrine differentiation, and absent or very low p64c-myc expression and the variant subclass with fast growth, absent to very low neuroendocrine differentiation, and high p64c-myc expression, we suggest a third subclass designated as transitional with moderate growth, moderate neuroendocrine differentiation, and high p64c-myc expression. Data on a small number of non-SCLC cell lines tested showed that high levels of p64c-myc correlate with high in vitro growth rates. This indicates that high p64c-myc levels may be associated with high proliferative activity, and lack of differentiation in lung cancer in general. The p74c-raf1 protein was found in all non-SCLC cell lines. Whether this classification of SCLC cell lines is applicable to SCLC in vivo remains to be determined.