A gene expression database for the molecular pharmacology of cancer.

We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

Identification of compounds with preferential inhibitory activity against low-Nm23-expressing human breast carcinoma and melanoma cell lines.

We have used the COMPARE computer algorithm and Nm23 expression as a marker of tumor metastatic potential to examine the in vitro antiproliferative activity of chemotherapeutic drugs on human breast carcinoma and melanoma cell lines. None of 171 compounds in clinical use or under development and only 40 of 30,000 repository compounds exhibited preferential growth inhibition of low-Nm23-expressing, metastatically aggressive cell lines with a Pearson correlation coefficient of < or = -0.64. Characterization of one compound, NSC 645306, is presented including in vivo activity in a hollow fiber assay. The data demonstrate a novel approach to drug identification for aggressive human tumors.

Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein.

Nucleocapsid p7 (NCp7) proteins of human immunodeficiency virus type 1 (HIV-1) contain two zinc binding domains of the sequence Cys-(X)2-Cys-(X)4-His-(X)4-Cys (CCHC). The spacing pattern and metal-chelating residues (3 Cys, 1 His) of these nucleocapside CCHC zinc fingers are highly conserved among retroviruses. These CCHC domains are required during both the early and late phases of retroviral replication, making them attractive targets for antiviral agents. toward that end, we have identified a number of antiviral chemotypes that electrophilically attack the sulfur atoms of the zinc-coordinating cysteine residues of the domains. Such nucleocapside inhibitors were directly virucidal by preventing the initiation of reverse transcription and blocked formation of infectious virus from cells through modification of CCHC domains within Gag precursors. Herein we report that azodicarbonamide (ADA) represents a new compound that inhibits HIV-1 and a broad range of retroviruses by targeting the the nucleocapsid CCHC domains. Vandevelde et al. also recently disclosed that ADA inhibits HIV-1 infection via an unidentified mechanism and that ADA was introduced into Phase I/II clinical trials in Europe for advanced AIDS. These studies distinguish ADA as the first known nucleocapsid inhibitor to progress to human trials and provide a lead compound for drug optimization.

An information-intensive approach to the molecular pharmacology of cancer.

Since 1990, the National Cancer Institute (NCI) has screened more than 60,000 compounds against a panel of 60 human cancer cell lines. The 50-percent growth-inhibitory concentration (GI50) for any single cell line is simply an index of cytotoxicity or cytostasis, but the patterns of 60 such GI50 values encode unexpectedly rich, detailed information on mechanisms of drug action and drug resistance. Each compound's pattern is like a fingerprint, essentially unique among the many billions of distinguishable possibilities. These activity patterns are being used in conjunction with molecular structural features of the tested agents to explore the NCI's database of more than 460,000 compounds, and they are providing insight into potential target molecules and modulators of activity in the 60 cell lines. For example, the information is being used to search for candidate anticancer drugs that are not dependent on intact p53 suppressor gene function for their activity. It remains to be seen how effective this information-intensive strategy will be at generating new clinically active agents.

Adaphostin and other anticancer drugs quench the fluorescence of mitochondrial potential probes.

Fluorescent dyes are widely used to monitor changes in mitochondrial transmembrane potential (DeltaPsim). When MitoTracker Red CMXRos, tetramethylrhodamine methyl ester (TMRM), and 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)) were utilized to examine the effects of the experimental anticancer drug adaphostin on intact cells or isolated mitochondria, decreased fluorescence was observed. In contrast, measurement of tetraphenylphosphonium uptake by the mitochondria using an ion-selective microelectrode failed to show any effect of adaphostin on DeltaPsim. Instead, further experiments demonstrated that adaphostin quenches the fluorescence of the mitochondrial dyes. Structure-activity analysis revealed that the adamantyl and p-aminobenzoic acid moieties of adaphostin are critical for this quenching. Anticancer drugs containing comparable structural motifs, including mitoxantrone, aminoflavone, and amsacrine, also quenched the mitochondrial probes. These results indicate the need for caution when mitochondrial dyes are utilized to examine the effects of xenobiotics on DeltaPsim and suggest that some previously reported direct effects of anticancer drugs on mitochondria might need re-evaluation.

Transcriptional activation and DNase I hypersensitive sites are associated with selective expression of the gastrin-releasing peptide gene.

The gastrin-releasing peptide (GRP) is a neuropeptide hormone and growth factor produced normally by neural and neuroendocrine cells, as well as by human small-cell lung cancer (SCLC) tumors and derived cell lines. This study compares the structure of the human prepro-GRP gene in four SCLC cell lines that express variable levels of steady-state GRP mRNA. The regulation of GRP gene expression appears to be at the level of primary transcription based on nuclear run on studies. In the two SCLC cell lines expressing GRP we find a single transcription start site for GRP mRNA, and near this site we find four DNase I hypersensitive sites. These hypersensitive sites are absent in the two cell lines that do not express GRP. The presence of DNase hypersensitive sites in the promoter region of the GRP gene is the structural feature that best correlates with transcriptional activation. These four DNase hypersensitive sites are candidates for cis acting regulatory regions, which may be important in determining the level of transcription of the human prepro GRP gene.

Human creatine kinase-B complementary DNA. Nucleotide sequence, gene expression in lung cancer, and chromosomal assignment to two distinct loci.

Using a small cell lung cancer (SCLC) cDNA library, we obtained clones for the creatine kinase-B (CK-B) gene and determined the nucleotide sequence for the protein coding and 3' untranslated region (3' UT). The human translated protein spans 381 residues and the amino acid homology with rabbit CK-B is greater than 98%. We have demonstrated that a nucleic acid probe encompassing the protein coding region will also hybridize to CK-M sequences while a probe derived from the 3' UT region is CK-B specific. When a B-isoenzyme specific sequence is hybridized to Eco RI cut genomic DNA, two independent restriction fragment polymorphisms are detected. We have subsequently localized these two CK-B homologous sequences to chromosomes 14q32 and 16. Finally, we show that increased levels of CK-B seen in SCLC are not accompanied by gene amplification or rearrangement, but reflect a greatly enhanced level of CK-B specific mRNA that is not seen in non-SCLC lines thus far examined.

Cholera toxin inhibits signal transduction by several mitogens and the in vitro growth of human small-cell lung cancer.

Cholera toxin (CT) inhibited the in vitro growth of three of four human small-cell lung carcinoma (SCLC) cell lines with a 50% inhibitory concentration of 27-242 ng/ml. Loss of surface membrane ruffling and the capacity of [Tyr4]-bombesin, vasopressin, and fetal calf serum to stimulate increases in intracellular free calcium clearly preceded effects on cellular metabolic activity and cell growth. 125I-[Tyr4]-bombesin binding was unaffected by CT treatment but [Tyr4]-bombesin stimulated phospholipase C activity was decreased in membranes from CT-treated SCLC cells. CT stimulated a rapid but transient increase in intracellular cyclic AMP ([cAMP]i) in SCLC. The effects of CT on susceptible SCLC were not reproduced by elevations of [cAMP]i induced by forskolin or cyclic AMP analogues. GM1 ganglioside, the cellular binding site for CT, was highly expressed in the CT-sensitive but not the CT-resistant SCLC cell lines. In contrast, expression of guanine nucleotide binding protein substrates for ADP-ribosylation by CT was similar. These data demonstrate the existence of a CT-sensitive growth inhibitory pathway in SCLC-bearing GM1 ganglioside. Addition of CT results in decreased responsiveness to several mitogenic stimuli. These results suggest novel therapeutic approaches to human SCLC.

Flavopiridol, a novel cyclin-dependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis.

Flavopiridol (HMR 1275) has been identified recently as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Flavopiridol inhibits most cyclin-dependent kinases (cdks) and displays unique anticancer properties. Here, we investigated whether this compound was effective against head and neck squamous cell carcinomas (HNSCC). Exposure of HNSCC cells to flavopiridol diminished cdc2 and cdk2 activity and potently inhibited cell proliferation (IC50 43-83 nM), which was concomitant with the appearance of cells with a sub-G1 DNA content. Moreover, DNA fragmentation and TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) reaction confirmed that flavopiridol induces apoptosis in all cell lines, even on certain HNSCC cells that are insensitive to apoptosis to DNA-damaging agents (gamma-irradiation and bleomycin). A tumorigenic HNSCC cell line was used to assess the effect of flavopiridol in vivo. Treatment (5 mg/kg per day, intraperitoneally) for 5 d led to the appearance of apoptotic cells in the tumor xenografts and caused a 60-70% reduction in tumor size, which was sustained over a period of 10 wk. Flavopiridol treatment also resulted in a remarkable reduction of cyclin D1 expression in HNSCC cells and tumor xenografts. Our data indicate that flavopiridol exerts antitumor activity in HNSCC, and thus it can be considered a suitable candidate drug for testing in the treatment of refractory carcinomas of the head and neck.

L-myc cooperates with ras to transform primary rat embryo fibroblasts.

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

Transcriptional inactivation of c-myc and the transferrin receptor in dibutyryl cyclic AMP-treated HL-60 cells.

Treatment of HL-60 cells with dibutyryl cyclic AMP induced rapid transcriptional inactivation of c-myc and the transferrin receptor. Transcriptional inactivation was followed by loss of c-myc and transferrin receptor mRNA and protein. Treated cells completed one round of proliferation, followed by growth arrest, G1 synchronization, and monocytic differentiation. These data suggest that cyclic AMP-mediated control of growth and differentiation may be achieved, at least in part, by transcriptional regulation of certain growth-associated proto-oncogenes and growth factor receptor genes.

Preclinical and clinical development of cyclin-dependent kinase modulators.

In the last decade, the discovery and cloning of the cyclin-dependent kinases (cdks), key regulators of cell cycle progression, have led to the identification of novel modulators of cdk activity. Initial experimental results demonstrated that these cdk modulators are able to block cell cycle progression, induce apoptotic cell death, promote differentiation, inhibit angiogenesis, and modulate transcription. Alteration of cdk activity may occur indirectly by affecting upstream pathways that regulate cdk activity or directly by targeting the cdk holoenzyme. Two direct cdk modulators, flavopiridol and UCN-01, are showing promising results in early clinical trials, in which the drugs reach plasma concentrations that can alter cdk activity in vitro. Although modulation of cdk activity is a well-grounded concept and new cdk modulators are being assessed for clinical testing, important scientific questions remain to be addressed. These questions include whether one or more cdks should be inhibited, how cdk inhibitors should be combined with other chemotherapy agents, and which cdk substrates should be used to assess the biologic effects of these drugs in patients. Thus, modulation of cdk activity is an attractive target for cancer chemotherapy, and several agents that modulate cdk activity are in or are approaching entry into clinical trials.

UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53.

BACKGROUND: Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G2 arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G2 checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent. PURPOSE: We studied the effect of UCN-01 on G2 checkpoint control in human lymphoma CA46 cells, whose sensitivity to various DNA-damaging agents and G2 response to DNA damage have been characterized. We also assessed the ability of UCN-01 to enhance the cytotoxicity of gamma irradiation in CA46 cells and human colon carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01-mediated abrogation of the G2 checkpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either expressed or did not express the human papillomavirus type-16 E6 protein. MCF-7 cells have normal p53 function, and the E6 protein binds p53 protein and promotes its destruction. METHODS: The effect of UCN-01 on cell cycle arrest induced by gamma irradiation was studied in CA46 cells and in transfected MCF-7 cells by use of flow cytometry. A histone H1 phosphorylation assay was employed to measure cyclin B1/Cdc2 kinase activity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphorylation status of Cdc2 kinase protein in the same extracts was determined by use of western blotting. The effect of UCN-01 on the cytotoxicity of gamma irradiation in CA46 and HT-29 cells was determined by use of MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respectively; a clonogenic assay was also used to measure the effect of UCN-01 on the cytotoxicity of cisplatin in transfected and nontransfected MCF-7 cells. RESULTS: G2 arrest induced in CA46 cells by gamma irradiation was minibited by treatment with UCN-01 in a dose-dependent manner; arrest in G2 was completely abrogated by exposure to 300 nM UCN-01. Biochemical markers indicative of the G2/M transition, including the activation of cyclin B1/Cdc2 kinase and the suppression of Cdc2 threonine-14 and tyrosine-15 phosphorylation, were detected in irradiated cells treated with UCN-01. UCN-01 enhanced the cytotoxicity of gamma irradiation in CA46 and HT-29 cells. MCF-7 cells with functional p53 protein were more resistant to G2 checkpoint abrogation by UCN-01 than MCF-7 cells with disrupted p53 function. UCN-01 markedly enhanced the cell-killing activity of cisplatin in MCF-7 cells defective for p53 function. CONCLUSIONS AND IMPLICATIONS: UCN-01 is a potent abrogator of G2 checkpoint control in cancer cells with disrupted p53 function. UCN-01 might be capable of enhancing the effectiveness of DNA-damaging agents in the treatment of tumors with cells lacking normal p53 function.

Jasplakinolide's inhibition of the growth of prostate carcinoma cells in vitro with disruption of the actin cytoskeleton.

BACKGROUND: Jasplakinolide, a cyclodepsipeptide produced by an Indo-Pacific sponge, Jaspis johnstoni, has been reported to inhibit the growth of breast cancer cells. PURPOSE: The effects of jasplakinolide on the proliferation of three human immortalized prostate carcinoma cell lines (PC-3, LNCaP, and TSU-Pr1) were studied. The growth-inhibitory effect of jasplakinolide on the PC-3 cell line was studied in detail to elucidate its mechanism of action. METHODS: Cell counts were used to study growth inhibition. A protein-based microplate assay was used to assess the time of exposure needed to cause persistent growth inhibition and to study the effects of jasplakinolide analogues. Metabolic changes were assessed by following the incorporation of radiolabeled precursors. The effects of jasplakinolide on the cytoskeleton were studied by fluorescent microscopy, using rhodamine phalloidin (RP) and antibodies to cytoskeletal components. Changes in RP binding were quantified by extracting bound fluorescent material from fixed cells and measuring the amount of fluorescence in a spectrofluorometer. RESULTS: The growth of PC-3, LNCaP, and TSU-Pr1 cells was potently inhibited by exposure to jasplakinolide for 48 hours; doses of jasplakinolide that led to 50% growth inhibition were 65 nM for PC-3 cells, 41 nM for LNCaP cells, and 170 nM for TSU-Pr1 cells. In PC-3 cells, exposure to 160 nM for 48 hours led to total growth inhibition, which persisted for several days even after drug removal. Several jasplakinolide analogues also inhibited the growth of PC-3 cells, although analogues in which the rigidity of the macrolide ring was altered were ineffective. No early changes in the synthesis of DNA, RNA, or protein or in intracellular adenosine triphosphate levels were seen in the PC-3 cells after exposure to jasplakinolide. Growth inhibition by jasplakinolide was accompanied by striking morphologic changes. Exposure for several doublings led to multinucleated cells. Further investigation of these changes in the PC-3 cells revealed a dramatic and early disruption of the actin cytoskeleton and a statistically significant decrease in RP binding. The doses of jasplakinolide, the time of exposure, and the pattern of growth inhibition by structural analogues corresponded with the changes seen in actin distribution. CONCLUSIONS: Jasplakinolide represents a novel marine natural product with potent in vitro antiproliferative activity against human prostate carcinoma cell lines, and it appears to target the actin cytoskeleton. IMPLICATIONS: Jasplakinolide is a potential candidate for further preclinical development and a lead structure for a novel class of therapeutic agents that can disrupt the actin cytoskeleton in mammalian cells.

Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines.

BACKGROUND: HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA. METHODS: We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided. RESULTS: The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA. CONCLUSIONS: Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.

Comparison of amplified and unamplified c-myc gene structure and expression in human small cell lung carcinoma cell lines.

A human small cell lung cancer cell line (H82) demonstrates 40- to 50-fold amplification of the c-myc gene but expresses at least 250-fold more steady-state c-myc messenger RNA than an unamplified small cell lung cancer cell line (H378) with no detectable expression of c-myc. We compared the chromatin structure of c-myc in H82 to that in H378 using DNase I sensitivity and DNA methylation patterns. DNase I hypersensitivity sites were identical in H82 and H378 and were similar to the pattern seen in a B-lymphoblastoid cell line, despite extensive amplification of c-myc in H82. Methylation patterns were also very similar in H82 and H378, with hypomethylation or partial methylation at the c-myc coding regions and the flanking 5' sequences, despite the absence of detectable c-myc expression in H378. Therefore, the predominant chromatin structural patterns do not appear to correlate with observed differences in gene expression. In addition, these studies demonstrate that the patterns of DNase I hypersensitivity and of methylation can remain intact during a 40- 50-fold gene amplification, as observed for the c-myc gene in H82.

Effect of guanine and adenine nucleotides on bombesin-stimulated phospholipase C activity in membranes from Swiss 3T3 and small cell lung carcinoma cells.

In [3H]inositol-labeled membranes prepared from Swiss mouse 3T3 and human small cell lung carcinoma cells, [Tyr4]-bombesin stimulated production of water-soluble inositol phosphates. The reaction was stimulated by guanosine 5'-O-[3-thiotriphosphate] and was specifically inhibited by both [Leu13-psi-CH2NHLeu14]-bombesin and the antibombesin antibody 2A11. [Tyr4]-bombesin-induced activation of phospholipase C is most apparent in Ca2(+)-depleted conditions (less than 1 microM[Ca2+]free). The kinetics of activation by ligand also demonstrate that [Tyr4]-bombesin-dependent phospholipase C activation is most apparent at [Mg2+]free of approximately 0.2 microM. At millimolar concentrations of [Mg2+]free, there is considerably less dependence on [Tyr4]-bombesin for activation of phospholipase C. ATP is not necessary for initial activation of phospholipase C, and beta, gamma-imidoadenosine-5'-triphosphate does not inhibit the reaction. These results demonstrate that in these cell types [Tyr4]-bombesin activates phospholipase C in conjunction with guanine nucleotides. Phospholipase C-coupled guanine nucleotide regulatory proteins would be appropriately considered as novel targets for the development of therapeutic strategies in small cell lung carcinoma.

Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells.

Flavopiridol (L86-8275), a N-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G1 or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cyclin D1 and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack CDK4-cyclin D1 to investigate the G1 arrest produced by Flavopiridol. Recombinant CDK4-cyclin D1 was inhibited potently by Flavopiridol (Kiapp, 65 nM), competitive with respect to ATP. Surprisingly, CDK4 immunoprecipitates derived from Flavopiridol-treated MCF-7 cells (3 h, 300 nM Flavonolpiridol) had an approximately 3-fold increased kinase activity compared with untreated cells. Cyclin D and CDK4 levels were not different at 3 hr, but cyclin D levels and CDK4 kinase activity decreased thereafter. The phosphorylation state of pRb was shifted from hypercoincident to hypocoincident with the development of G1 arrest. Asynchronous MDA-MB-468 cells were inhibited in cell cycle progression at both G1 and G2 by Flavopiridol. Flavopiridol inhibited the in vitro kinase activity of CDK2 using an immune complex kinase assay (IC50, 100 nM at 400 microM ATP). Immunoprecipitated CDK2 kinase activity from either MCF-7 or MDA-MB-468 cells exposed to Flavopiridol (300 nM) for increasing time showed an initial increased activity (approximately 1.5-fold at 3 h) compared with untreated cells, followed by a loss of kinase activity to immeasurable levels by 24 h. This increased immunoprecipitated kinase activity was dependent on the Flavopiridol concentration added to intact cells and was associated with a reduction of CDK2 tyrosine phosphorylation. Cyclin E and A levels were not altered to the same extent as cyclin D, and neither CDK4 nor CDK2 levels were changed in response to Flavopiridol. Inhibition of the CDK4 and/or CDK2 kinase activity by Flavopiridol can therefore account for the G1 arrest observed after exposure to Flavopiridol.

Growth inhibition by cholera toxin of human lung carcinoma cell lines: correlation with GM1 ganglioside expression.

The effect of cholera toxin (CT) on the growth of 12 small cell lung carcinoma (SCLC) and 15 non-small cell lung carcinoma (NSCLC) cell lines is presented. CT inhibited the growth of nine SCLC cell lines (concentration for 50% inhibition of growth, 27-700 ng/ml), all of which had abundant expression of GM1 ganglioside, the surface receptor for CT. CT-resistant SCLC all had greatly decreased GM1 expression. In contrast, CT inhibited the growth of only four of 15 NSCLC cell lines. Seven of the 11 CT-resistant NSCLC had levels of GM1 comparable to CT-sensitive NSCLC or SCLC. In a limited panel of cell lines, cyclic AMP (cAMP) agonists including forskolin, 8Br[cAMP], and dibutyryl[cAMP] did not consistently reproduce CT-mediated inhibition of cell growth, nor did these compounds overcome resistance of cells to the growth inhibitory effects of CT. Expression of the RI and RII regulatory subunits of cAMP-dependent protein kinase was similar in CT-resistant and CT-sensitive SCLC or NSCLC cell lines. In the presence of isobutylmethylxanthine, intracellular cAMP levels induced by CT in a CT-resistant, GM1(+) NSCLC cell line were comparable to those achieved in a CT-sensitive NSCLC cell line. We conclude that inhibition of lung carcinoma cell growth by CT in all cases requires expression of GM1, and in the case of SCLC cell lines the presence of GM1 is sufficient. In NSCLC cell lines, expression of GM1 is not sufficient for growth inhibition by CT. These findings imply refractoriness to growth inhibition by cAMP in GM1(+), CT-resistant NSCLC cell lines and the possibility of non-cAMP-related mechanisms for growth inhibition in CT-sensitive cell lines.

Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells.

UCN-01 is a derivative of staurosporine, initially developed as a potentially selective inhibitor of the Ca(2+)- and phospholipid-dependent protein kinase C, but with the capacity to inhibit a number of tyrosine and serine/threonine kinases. UCN-01 inhibits the growth of 5 breast carcinoma cell lines with a 50% inhibitory concentration range of 30-100 nM during 6 days of continuous exposure. In MCF-7, MDA-MB453, and SK-BR-3 cells, UCN-01 is 5-fold more potent in growth inhibition than its diastereomer UCN-02, but the 2 compounds are equipotent in the inhibition of MDA-MB468 and H85787 cell growth. A differential sensitivity to a 24-h period of exposure to UCN-01 followed by drug removal and growth for 5 subsequent days was observed. The rank order for persistent inhibition of cells by UCN-01 was MCF-7, MDA-MB453 >> SK-BR-3 > H85787 > MDA-MB468. MCF-7 and MDA-MB453 cells did not resume proliferation within the 5 days after brief exposure to UCN-01. In contrast, MDA-MB468 and H85787 cells showed no net growth inhibition after a 24-h pulse of UCN-01, followed by 5 more days of growth in drug-free medium. In MDA-MB468 cells, 150 nM UCN-01 retards but does not prevent cell cycle progression through S phase, but the cells are clearly blocked from exit of G1 and entry into S. Progression through S phase is completely inhibited by 600 nM UCN-01. The development of a G1 to S block by UCN-01 in MDA-MB468 cells occurs in conjunction with inhibition of [32P]orthophosphate labeling and decreased phosphotyrosine mass of discrete cellular phosphoproteins.