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.

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.

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.

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.

Multiple mechanisms for transcriptional regulation of the myc gene family in small-cell lung cancer.

The molecular mechanisms reported to regulate the expression of myc family genes are multiple and complex and include gene amplification, transcriptional activation, transcriptional attenuation, and mRNA stability. We have investigated which of these mechanisms are responsible for the extreme variation in myc gene family mRNA levels observed in human small-cell lung cancer cell lines. In addition to gene amplification, a block to nascent mRNA chain elongation, causing attenuation of transcription, is an important regulatory mechanism controlling the steady-state levels of c-myc and L-myc mRNA. The loss of transcriptional attenuation is correlated with overexpression of these two genes in cell lines which do not show gene amplification. Expression of c-myc mRNA appears to be dependent on promoter activity and attenuator function. In contrast, regulation of expression of the N-myc gene does not involve transcriptional attenuation; steady-state mRNA levels are correlated with promoter activity as well as gene amplification. We conclude that transcriptional regulation of each member of the myc gene family is accomplished by a different assortment of complex mechanisms, including gene copy number, promoter activation, and transcriptional attenuation. Interference at multiple points in this complex regulatory process appears to be an important mechanism by which small-cell lung cancer and other human tumors evade growth control.

The human L-myc gene encodes multiple nuclear phosphoproteins from alternatively processed mRNAs.

The human proto-oncogene L-myc generates at least four different mRNAs by alternative RNA processing. We have identified two phosphorylated L-myc proteins with molecular masses of 60,000 and 66,000 daltons [p60L-myc(human) and p66L-myc(human)] in a small-cell carcinoma line expressing high levels of L-myc mRNA. These proteins have a short half-life and are localized to the nuclear matrix fraction, as previously reported for the c-myc and N-myc proteins. In vitro translation experiments demonstrated that both the p60 and p66 species are encoded by a 3.9-kilobase (kb) mRNA which retains intron 1, while only the p60 protein is translated from a 3.6-kb L-myc mRNA which has had intron 1 removed. While L-myc proteins [p32L-myc(human) and p37L-myc(human)] could be synthesized in vitro from 2.2-kb mRNA templates, no such proteins were detected by immunoprecipitation in vivo. These observations suggest that alternative RNA processing of the L-myc transcript could play a role in determining the steady-state levels of the p60L-myc and p66L-myc proteins.

Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing.

We analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs there is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

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.

Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275.

BACKGROUND: Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can inhibit tumor cell growth in vitro, and preliminary in vivo studies of the flavone L86-8275 have shown growth inhibition of LX529 and A549 lung carcinomas. L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] is a flavone of novel structure. PURPOSE: The purpose of this study was to determine in vitro whether L86-8275 is a more potent inhibitor of growth in breast carcinoma and lung carcinoma cells than quercetin or genistein. METHODS: We studied the effects of L86-8275 on cell growth in seven breast carcinoma cell lines and five lung carcinoma cell lines. MDA468 breast carcinoma was then selected for further study. Cell proliferation was measured by a colorimetric dye reduction assay; synthesis of DNA, RNA, and protein by incorporation of the radioactive metabolic precursors thymidine, uridine, or leucine, respectively; adenosine triphosphate (ATP) content by a luciferase-mediated bioluminescence reaction; and cell cycle progression by the use of cell-synchronizing drugs (aphidicolin and nocodazole) and flow cytometry. RESULTS: L86-8275 was not cytotoxic to stationary-phase cells but reversibly inhibited the growth of cells in exponential growth phase. At concentrations of 25-160 nM, L86-8275 inhibited growth of human breast and lung carcinoma cell lines by 50%. MDA468 breast carcinoma cells were 60-fold and 400-fold more sensitive to L86-8275 than to quercetin and genistein, respectively. By 24 hours after addition of L86-8275, DNA synthesis in MDA468 cells was inhibited by greater than 95%, protein synthesis by 80%, and RNA synthesis by 40%-60%, under conditions that preserved cellular ATP levels at approximately 80%-90% of control values. When MDA468 cells released from aphidicolin-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed the S phase but arrested in G2. When cells released from nocodazole-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed mitosis but arrested in G1. CONCLUSIONS: L86-8275 is a potent, yet reversible, growth-inhibitory flavone that can selectively block cell cycle progression in vitro at more than one point in the cell cycle. IMPLICATIONS: These findings suggest that L86-8275 is a candidate for further preclinical development, as well as a model for the synthesis of other flavonoids that might potently delay cell cycle progression to achieve inhibition of tumor growth. Future studies need to address optimal schedules for antiproliferative activity in vivo and inhibition of clonogenic activity.

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.