CHEK2 genomic and proteomic analyses reveal genetic inactivation or endogenous activation across the 60 cell lines of the US National Cancer Institute.

CHEK2 encodes a serine/threonine kinase (Chk2) activated by ATM in response to DNA double-strand breaks. On the one hand, CHEK2 has been described as a tumor suppressor with proapoptotic, cell-cycle checkpoint and mitotic functions. On the other hand, Chk2 is also commonly activated (phosphorylated at T68) in cancers and precancerous lesions. Here, we report an extensive characterization of CHEK2 across the panel of 60 established cancer cell lines from the NCI Anticancer Screen (the NCI-60) using genomic and proteomic analyses, including exon-specific mRNA expression, DNA copy-number variation (CNV) by aCGH, exome sequencing, as well as western blot analyses for total and activated (pT68-Chk2) Chk2. We show that the high heterogeneity of Chk2 levels in cancer cells is primarily due to its inactivation (owing to low gene expression, alternative splicing, point mutations, copy-number alterations and premature truncation) or reduction of protein levels. Moreover, we observe that a significant percentage of cancer cells (12% of the NCI-60 and HeLa cells) show high endogenous Chk2 activation, which is always associated with p53 inactivation, and which is accompanied by downregulation of the Fanconi anemia and homologous recombination pathways. We also report the presence of activated Chk2 (pT68-Chk2) along with histone γ-H2AX in centrosomes.

Increased expression of beta 2-microglobulin in multidrug-resistant tumour cells.

The rat monoclonal antibody LMR-12 was shown earlier to react with a plasma membrane protein, upregulated in multidrug-resistant cell lines. In this study, we observed distinct LMR-12 staining in 36 out of 55 non-drug-selected tumour cell lines, including melanomas, renal cell-, colon- and lung carcinomas, whereas in other tumour types, such as leukaemia and ovarian cancer, LMR-12 staining was generally low or absent. The cDNA encoding the LMR-12 antigen was isolated from a library of the multidrug-resistant human fibrosarcoma cell line HT1080/DR4 by expression cloning in MOP8 cells. Sequence analysis showed that the LMR-12 antigen is identical to the major histocompatibility complex class I molecule beta 2-microglobulin (beta2-m). The LMR-12/ beta2-m staining results were confirmed by mRNA microarray data from an independent National Cancer Institute study, as well as by newly obtained reverse transcriptase polymerase chain reaction data. Further analysis of the microarray data showed that beta2-m levels closely reflected levels of major histocompatibility complex class I heavy chains and the transporter associated with antigen processing. Since the ABC transporter associated with antigen processing was previously shown to contribute to multidrug-resistance, it may very well be that the observed LMR-12/ beta2-m levels are secondary to (elevated) levels of the transporter associated with antigen processing. A perspective arising from the present study is that drug resistant tumour cells may, by having elevated levels of major histocompatibility complex related molecules, be particular good candidates for alternative therapeutic therapies, such as cytotoxic T cell mediated immune-therapies.

Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.

BACKGROUND: Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated. RESULTS: Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover. CONCLUSIONS: The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

Expression and potential role of Fas-associated phosphatase-1 in ovarian cancer.

Fas-associated phosphatase-1 (FAP-1) is a protein-tyrosine phosphatase that binds the cytosolic tail of Fas (Apo1, CD95), presumably regulating Fas-induced apoptosis. Elevations of FAP-1 protein levels in some tumor cell lines have been correlated with resistance to Fas-induced apoptosis. To explore the expression of FAP-1 in ovarian cancer cell lines and archival tumor specimens, mouse monoclonal and rabbit polyclonal antibodies were generated against a FAP-1 peptide and recombinant FAP-1 protein. These antibodies were used for immunoblotting, immunohistochemistry, and flow-cytometry analysis of FAP-1 expression in the Fas-sensitive ovarian cancer lines HEY and BG-1, and in the Fas-resistant lines OVCAR-3 FR and SK-OV-3. All methods demonstrated high levels of FAP-1 in the resistant lines OVCAR-3 FR and SK-OV-3, but not in the Fas-sensitive lines HEY and BG-1. Furthermore, levels of FAP-1 protein also correlated with the amounts of FAP-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction analysis. FAP-1 protein levels were investigated by immunoblotting in the National Cancer Institute's panel of 60 human tumor cell lines. Although FAP-1 failed to correlate with Fas-resistance across the entire tumor panel, Fas-resistance correlated significantly with FAP-1 expression (P: < or = 0.05) and a low Fas/FAP-1 ratio (P: < or = 0.028) in ovarian cancer cell lines. FAP-1 expression was also evaluated in 95 archival ovarian cancer specimens using tissue-microarray technology. FAP-1 was expressed in nearly all tumors, regardless of histological type or grade, stage, patient age, response to chemotherapy, or patient survival. We conclude that FAP-1 correlates significantly with Fas resistance in ovarian cancer cell lines and is commonly expressed in ovarian cancers.

Thymidine kinase, thymidylate synthase, and dihydropyrimidine dehydrogenase profiles of cell lines of the National Cancer Institute's Anticancer Drug Screen.

PURPOSE: To determine the expression of three targets of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd) in human tumor cell lines and to compare these with the 50% growth inhibition concentrations (GI(50)) from the National Cancer Institute database. EXPERIMENTAL DESIGN: Thymidine kinase (TK) activity was assessed by conversion of [(3)H]thymidine to [(3)H]TMP. Thymidylate synthase (TS) protein expression was determined by Western analysis. TS and dihydropyrimidine dehydrogenase (DPD) mRNA expression were measured by quantitative reverse transcription-PCR. RESULTS: The median (range) for the targets were as follows: 5-FU GI(50), 20.8 microM (0.8-536); FdUrd GI(50), 0.75 microM (0.25-237); TK, 0.93 nmol/min/mg (0.16-5.7); in arbitrary units: TS protein, 0.41 (0.05-2.95); TS mRNA, 1.05 (0.12-6.41); and DPD mRNA, 1.09 (0.00-24.4). A moderately strong correlation was noted between 5-FU and FdUrd GI(50)s (r = 0.60), whereas a weak-moderate correlation was seen between TS mRNA and protein expression (r = 0.45). Neither TS expression nor TK activity correlated with 5-FU or FdUrd GI(50)s, whereas lines with lower DPD expression tended to be more sensitive to 5-FU. Cell lines with faster doubling times and wild-type p53 were significantly more sensitive to 5-FU and FDURD: CONCLUSIONS: The lack of correlation may in part be attributable to the influence of downstream factors such as p53, the observation that the more sensitive cell lines with faster doubling times also had higher TS levels, and the standard procedure of the screen that uses a relatively short (48-h) drug exposure.

P450 enzyme expression patterns in the NCI human tumor cell line panel.

Cytochrome P450 (P450) enzyme expression patterns were determined for a panel of 60 human tumor cell lines, representing nine tumor tissue types, used by the National Cancer Institute (NCI) Anticancer Drug Screening Program. All 60 tumor cell lines displayed significant P450 activity, as well as P450 reductase activity, as determined using the general P450 substrate 7-benzyloxyresorufin. Cell line-specific P450 enzyme patterns were observed using three other P450 substrates, 7-ethoxycoumarin, coumarin, and 7-ethoxyresorufin, each of which was metabolized at a low rate. Using a pattern-matching computer program, COMPARE, correlative relationships were investigated between the arrays of P450 activities and the patterns of cytotoxicity exhibited by a large group of anticancer agents of proven or potential clinical utility. Significant negative correlations between the patterns of P450-dependent 7-benzyloxyresorufin metabolism activity and cell line chemosensitivity were observed for 10 standard anticancer agents (including 6 alkylating agents) and 55 investigational compounds, suggesting a role for P450 metabolism in the inactivation of these agents. Negative correlations between 7-ethoxycoumarin O-deethylation and cell line chemosensitivity to a group of topoisomerase inhibitors were also seen, again suggesting P450-dependent drug inactivation. P450 enzyme profiling may thus aid in interpreting the patterns of drug sensitivity and resistance in the NCI tumor cell panel, and may facilitate the identification of anticancer agents whose activity can be altered via cytochrome P450 metabolism.

Modulation of the multidrug-resistance phenotype by new tropane alkaloid aromatic esters from Erythroxylum pervillei.

Nine tropane alkaloid aromatic esters (1-9) were isolated from the roots of Erythroxylum pervillei by following their potential to reverse multidrug-resistance with vinblastine-resistant oral epidermoid carcinoma (KB-V1) cells. All isolates, including seven new structures (3-9), were evaluated against a panel of human cancer cell lines, and it was found that alkaloids 3 and 5-9 showed the greatest activity with KB-V1 cells assessed in the presence of vinblastine, suggesting that these new compounds are potent modulators of P-glycoprotein. Confirmatory results were obtained with human ovarian adenocarcinoma (SKVLB) cells evaluated in the presence of adriamycin and synergistic studies performed with several cell lines from the NCI tumor panel. The structures of the new compounds were determined using spectroscopic techniques. Single-crystal X-ray analysis was performed on the monoester, tropane-3 alpha,6 beta,7 beta-triol 3-phenylacetate (1).

TNFR-associated factor family protein expression in normal tissues and lymphoid malignancies.

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.

UCN-01 enhances the in vitro toxicity of clinical agents in human tumor cell lines.

UCN-01 is undergoing Phase I evaluation and is a candidate for combination strategies in the clinic. UCN-01 has been shown to have a variety of effects on cellular targets and the cell cycle. It has also been reported to sensitize cells to several clinical drugs in vitro, possibly in a manner related to p53 status. Thus, combinations of UCN-01 with a series of clinical agents in variety of cell lines have been investigated in vitro. Certain cell lines demonstrated synergistic interactions with combinations of UCN-01 (20-150 nM) and thiotepa, mitomycin C, cisplatin, melphalan, topotecan, gemcitabine, fludarabine or 5-fluorouracil. In contrast, UCN-01 combinations with the antimitotic agents, paclitaxel and vincristine, or topoisomerase II inhibitors, adriamycin and etoposide, did not result in synergy, only in additive toxicity. Cells with non-functional p53 were significantly more susceptible to the supra-additive effects of certain DNA-damaging agents and UCN-01 combinations, than cells expressing functional p53 activity. In contrast, there was no significant relationship between p53 status and susceptibility to synergy between antimetabolites and UCN-01. The mechanism behind the observed synergy appeared unrelated to effects on protein kinase C or abrogation of the cell cycle in G2. Moreover, increased apoptosis did not fully explain the supradditive response. These data indicate that UCN-01 sensitizes a variety of cell lines to certain DNA-damaging agents (frequently covalent DNA-binding drugs) and antimetabolites in vitro, but the mechanism underlying this interaction remains undefined.

Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias.

Expression of several inhibitor of apoptosis proteins (IAPs) was investigated in the National Cancer Institute panel of 60 human tumor cell lines, and the expression and prognostic significance of one of these, XIAP, was evaluated in 78 previously untreated patients with acute myelogenous leukemia (AML). XIAP and cIAP1 were expressed in most cancer lines analyzed, with substantial variability in their relative levels. In contrast, NAIP mRNA was not detectable, and cIAP2 was found at the mRNA and protein levels in only 34 (56%) and 5 (8%) of the 60 tumor cell lines analyzed, respectively. Interestingly, XIAP, cIAP1, and cIAP2 mRNA levels did not correlate with protein levels in the tumor lines, indicating posttranscriptional regulation of expression. High levels of XIAP protein in tumor cell lines were unexpectedly correlated with sensitivity to some anticancer drugs, particularly cytarabine and other nucleosides, whereas higher levels of cIAP1 protein levels were associated with resistance to several anticancer drugs. The relevance of XIAP to in vivo responses to cytarabine was explored in AML, making correlations with patient outcome (n = 78). Patients with lower levels of XIAP protein had significantly longer survival (median, 133 versus 52.5 weeks; P = 0.05) and a tendency toward longer remission duration (median, 87 versus 52.5 weeks; P = 0.13) than those with higher levels of XIAP. Altogether, these findings show that IAPs are widely but differentially expressed in human cancers and leukemias and suggest that higher XIAP protein levels may have adverse prognostic significance for patients with AML.

The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network.

The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), have been implicated in human tumor development and metastasis. HGF/SF induces the expression of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), important mediators of cell invasion and metastasis. We have developed a cell-based assay to screen for inhibitors of this signaling system using the induction of endogenous uPA and uPAR and the subsequent conversion of plasminogen to plasmin as the biological end point. Assay validation was established using a neutralizing antiserum to HGF/SF and a uPA inhibitor (B428), as well as inhibitors of the MKK-MAPK1/2 pathway, shown previously to be important in the induction of uPA and uPAR. Using this assay, we found several classes of molecules that exhibited inhibition of HGF/SF-dependent plasmin activation. However, we discovered that certain members of the geldanamycin family of anisamycin antibiotics are potent inhibitors of HGF/SF-mediated plasmin activation, displaying inhibitory properties at femtomolar concentrations and nine orders of magnitude below their growth inhibitory concentrations. At nanomolar concentrations, the geldanamycins down-regulate Met protein expression, inhibit HGF/SF-mediated cell motility and invasion, and also revert the phenotype of both autocrine HGF/SF-Met transformed cells as well as those transformed by Met proteins with activating mutations. Thus, the geldanamycins may have important therapeutic potential for the treatment of cancers in which Met activity contributes to the invasive/metastatic phenotype.

Modulation of clinical drug resistance in a B cell lymphoma patient by the protein kinase inhibitor 7-hydroxystaurosporine: presentation of a novel therapeutic paradigm.

Emerging evidence suggests that apoptosis is an important mechanism of tumor cell death from antineoplastic therapy. 7-hydroxystaurosporine (UCN-01) is a novel protein kinase inhibitor that increases chemotherapy-induced apoptosis in vitro and is in early phases of clinical development. In this report, we present a 68-year-old patient with chemotherapy-resistant lymphoma treated with UCN-01 and chemotherapy. He had a stage IV plasmacytoid lymphoma that failed to enter remission with high-dose EPOCH II (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin) chemotherapy. Due to disease progression and transformation to large cell lymphoma in the liver and bone marrow, he received UCN-01. Four weeks later, he received "standard-dose" EPOCH because of progression, developed severe neutropenia for 9 days, and expired from Candida sepsis on day 23. At autopsy, there was no histological evidence of residual lymphoma, although PCR for immunoglobulin gene rearrangement analysis revealed a faint clonal band in two of six nodes but none in the liver. Significantly, no B cells were detected by immunohistochemistry in lymph nodes, and a polyclonal ladder pattern associated with the presence of normal B cells was not seen in the immunoglobulin gene rearrangement PCR assay. Profound peripheral lymphopenia (50 cells/microliter) was also observed. Pharmacokinetics showed UCN-01 salivary concentrations, a surrogate for free drug concentrations, to be within an effective range in vitro (45 nmol/L) as a modulator of DNA-damaging agent cytotoxicity. In vitro, UCN-01 is synergistic with multiple cytotoxic agents and increases fludarabine-induced apoptosis in a human breast cell line. These results suggest that UCN-01 sensitized the lymphoma to the cytotoxic effects of EPOCH, possibly by modulating the "threshold" for apoptosis, and may illustrate a new paradigm for reversal of drug resistance.

Characterization of MLH1 and MSH2 DNA mismatch repair proteins in cell lines of the NCI anticancer drug screen.

PURPOSE AND METHODS: The lack of a functional DNA mismatch repair (MMR) pathway has been recognized as a common characteristic of several different types of human cancers due to mutation affecting one of the MMR genes or due to promoter methylation gene silencing. These MMR-deficient cancers are frequently resistant to alkylating agent chemotherapy such as DNA-methylating or platinum-containing compounds. To correlate drug resistance with MMR status in a large panel of human tumor cell lines, we evaluated by Western blot the cellular levels of the two MMR proteins most commonly mutated in human cancers, MLH1 and MSH2, in the NCI human tumor cell line panel. This panel consists of 60 cell lines distributed among nine different neoplastic diseases. RESULTS: We found that in most of these cell lines both MLH1 and MSH2 were expressed, although at variable levels. Five cell lines (leukemia CCRF-CEM, colon HCT 116 and KM12 and ovarian cancers SK-OV-3 and IGROV-1) showed complete deficiency in MLH1 protein. MSH2 protein was detected in all 57 cell lines studied. Absence of MLH1 protein was always linked to resistance to the methylating chemotherapeutic agent temozolomide. This resistance was independent of cellular levels of O6-alkylguanine DNA alkyltransferase. Based on data available for review in the NCI COMPARE database, cellular levels of MLH1 and MSH2 did not correlate significantly with sensitivity to any standard anticancer drug or with any characterized molecular target already tested against the same panel of cell lines. CONCLUSION: Based on evaluation of 60 tumor cell lines in the NCI anticancer drug screen, MLH1 deficiency was more common than MSH2 deficiency and was always associated with a high degree of temozolomide resistance. These data will enable correlations with other drug sensitivities and molecular targets in the COMPARE database to evaluate linked processes in tumor drug resistance.

Interaction of the P-glycoprotein multidrug transporter (MDR1) with high affinity peptide chemosensitizers in isolated membranes, reconstituted systems, and intact cells.

P-glycoprotein-mediated multidrug resistance can be reversed by the action of a group of compounds known as chemosensitizers. The interactions with P-glycoprotein of two novel hydrophobic peptide chemosensitizers (reversins 121 and 205) have been studied in model systems in vitro, and in a variety of MDR1-expressing intact tumor cells. The reversins bound to purified P-glycoprotein with high affinity (77-154 nM), as assessed by a quenching assay using fluorescently labeled purified protein. The peptides modulated P-glycoprotein ATPase activity in Sf9 insect cell membranes expressing human MDR1, plasma membrane vesicles from multidrug-resistant cells, and reconstituted proteoliposomes. Both peptides induced a large stimulation of ATPase activity; however, higher concentrations, especially of reversin 205, led to inhibition. This pattern was different from that of simple linear peptides, and resembled that of chemosensitizers such as verapamil. In both membrane vesicles and reconstituted proteoliposomes, 1-2 microM reversins were more effective than cyclosporin A at blocking colchicine transport. Reversin 121 and reversin 205 restored the uptake of [3H]daunorubicin and rhodamine 123 in MDR1-expressing cells to the level observed in the drug-sensitive parent cell lines, and also effectively inhibited the extrusion of calcein acetoxymethyl ester from intact cells. In cytotoxicity assays, reversin 121 and reversin 205 eliminated the resistance of MDR1-expressing tumor cells against MDR1-substrate anticancer drugs, and they had no toxic effects in MDR1-negative control cells. We suggest that peptides of the reversin type interact with the MDR1 protein with high affinity and specificity, and thus they may be good candidates for the development of MDR1-modulating agents to sensitize drug resistance in cancer.

The ras oncogene-mediated sensitization of human cells to topoisomerase II inhibitor-induced apoptosis.

BACKGROUND: Among the inhibitors of the enzyme topoisomerase II (an important target for chemotherapeutic drugs) tested in the National Cancer Institute's In Vitro Antineoplastic Drug Screen, NSC 284682 (3'-hydroxydaunorubicin) and NSC 659687 [9-hydroxy-5,6-dimethyl-1-(N-[2(dimethylamino)ethyl]carbamoyl)-6H-pyrido -(4,3-b)carbazole] were the only compounds that were more cytotoxic to tumor cells harboring an activated ras oncogene than to tumor cells bearing wild-type ras alleles. Expression of the multidrug resistance proteins P-glycoprotein and MRP (multidrug resistance-associated protein) facilitates tumor cell resistance to topoisomerase II inhibitors. We investigated whether tumor cells with activated ras oncogenes showed enhanced sensitivity to other topoisomerase II inhibitors in the absence of the multidrug-resistant phenotype. METHODS: We studied 20 topoisomerase II inhibitors and individual cell lines with or without activated ras oncogenes and with varying degrees of multidrug resistance. RESULTS: In the absence of multidrug resistance, human tumor cell lines with activated ras oncogenes were uniformly more sensitive to most topoisomerase II inhibitors than were cell lines containing wild-type ras alleles. The compounds NSC 284682 and NSC 659687 were especially effective irrespective of the multidrug resistant phenotype. The ras oncogene-mediated sensitization to topoisomerase II inhibitors was far more prominent with the non-DNA-intercalating epipodophyllotoxins than with the DNA-intercalating inhibitors. This difference in sensitization appears to be related to a difference in apoptotic sensitivity, since the level of DNA damage generated by etoposide (an epipodophyllotoxin derivative) in immortalized human kidney epithelial cells expressing an activated ras oncogene was similar to that in the parental cells, but apoptosis was enhanced only in the former cells. CONCLUSIONS: Activated ras oncogenes appear to enhance the sensitivity of human tumor cells to topoisomerase II inhibitors by potentiating an apoptotic response. Epipodophyllotoxin-derived topoisomerase II inhibitors should be more effective than the DNA-intercalating inhibitors against tumor cells with activated ras oncogenes.

Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents.

In the present study, we report the characterization of the p53 tumor suppressor pathway in the 60 cell lines of the National Cancer Institute (NCI) anticancer drug screen, as well as correlations between the integrity of this pathway and the growth-inhibitory potency of 123 anticancer agents in this screen. Assessment of p53 status in these lines was achieved through complete p53 cDNA sequencing, measurement of basal p53 protein levels and functional assessment of (a) transcriptional activity of p53 cDNA from each line in a yeast assay, (b) gamma-ray-induced G1 phase cell cycle arrest, and (c) gamma-ray-induced expression of CIP1/WAF1, GADD45, and MDM2 mRNA. Our investigations revealed that p53 gene mutations were common in the NCI cell screen lines: 39 of 58 cell lines analyzed contained a mutant p53 sequence. cDNA derived from almost all of the mutant p53 cell lines failed to transcriptionally activate a reporter gene in yeast, and the majority of mutant p53 lines studied expressed elevated basal levels of the mutant p53 protein. In contrast to most of the wild-type p53-containing lines, cells containing mutant p53 sequence were also deficient in gamma-ray induction of CIP1/WAF1, GADD45, and MDM2 mRNA and the ability to arrest in G1 following gamma-irradiation. Taken together, these assessments provided indications of the integrity of the p53 pathway in the 60 cell lines of the NCI cell screen. These individual p53 assessments were subsequently used to probe a database of growth-inhibitory potency for 123 "standard agents," which included the majority of clinically approved anticancer drugs. These 123 agents have been tested against these lines on multiple occasions, and a proposed mechanism of drug action had previously been assigned to each agent. Our analysis revealed that cells with mutant p53 sequence tended to exhibit less growth inhibition in this screen than the wild-type p53 cell lines when treated with the majority of clinically used anticancer agents: including DNA cross-linking agents, antimetabolites, and topoisomerase I and II inhibitors. Similar correlations were uncovered when we probed this database using most of the other indices of p53 status we assessed in the lines. Interestingly, a class of agents that differed in this respect was the antimitotic agents. Growth-inhibitory activity of these agents tended, in this assay, to be independent of p53 status. Our characterization of the p53 pathway in the NCI cell screen lines should prove useful to researchers investigating fundamental aspects of p53 biology and pharmacology. This information also allows for the large-scale analysis of the more than 60,000 compounds tested against these lines for novel agents that might exploit defective p53 function as a means of preferential toxicity.

Resistance to paclitaxel mediated by P-glycoprotein can be modulated by changes in the schedule of administration.

PURPOSE: Increasing use of paclitaxel in clinical oncology has stimulated interest in its mechanisms of resistance and ways to overcome these. Studies were performed with paclitaxel to determine the role of P-glycoprotein in drug sensitivity, and the effect of schedule on relative resistance. We have previously reported that prolonged exposure to P-glycoprotein substrates decreases relative resistance in multidrug resistant cells. METHODS: Using both unselected and drug-selected cell lines, cross-resistance and cytotoxicity reversal studies using cyclosporin A were performed. In multidrug-resistant cells, cross-resistance was evaluated after 3-, 24-, and 96-h exposures to paclitaxel. RESULTS: Cross-resistance to paclitaxel in P-glycoprotein-expressing sublines was shown to be comparable to that of other drugs transported by P-glycoprotein. Sensitivity to paclitaxel could be modulated by cyclosporin A in unselected cell lines expressing P-glycoprotein and not in P-glycoprotein-negative cell lines. Resistance to paclitaxel was reduced tenfold by increasing the duration of exposure in P-glycoprotein-expressing cells. This effect was not observed in a paclitaxel-resistant cell line which does not express P-glycoprotein. CONCLUSIONS: These studies extend observations on the schedule dependence of paclitaxel cytotoxicity and the role of P-glycoprotein in mediating paclitaxel sensitivity. The schedule dependence of relative resistance suggests that infusional paclitaxel may help in overcoming P-glycoprotein-mediated resistance.

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.