The experience with voluntary genomic data submissions at the FDA and a vision for the future of the voluntary data submission program.

Drug developers have been using genomic information in drug development strategies for a number of years, but it was unclear how this information would be reviewed by the Food and Drug Administration (FDA). In order to evaluate the regulatory impact of genomic data in current drug development, a workshop was held in May 2002 to discuss aspects surrounding genomic data submission to the FDA (Figure 1).

Large-scale gene expression analysis in molecular target discovery.

The evolution of simple arrays consisting of a few genes to ones composed of thousands of genes and/or ESTs has allowed investigators unprecedented views of the molecular mechanisms within cells. Due to the enormous quantities of information derived from microarray analysis, new types of problems have surfaced, such as where to store all of the data. The ability to solve database or statistical problems has required the bench biologist to collaborate with database developers, software designers and statisticians to determine solutions for storage, analysis and interpretation of microarray data. The collaborative effort between these extremely diverse disciplines has led to the development of creative database query and gene expression analysis tools, producing significant reductions in the time required by researchers to filter through the datasets and discover the key processes perturbed by the diseases of interest. Both unsupervised and supervised analysis methods have been applied to gene expression data leading to the discovery of novel therapeutic targets and diagnostic markers. Furthermore, tumor classification based on their respective molecular fingerprints has led to the classification of cancer subtypes and the discovery of novel molecular taxonomies that may eventually lead to improved patient stratification and superior therapeutic strategies.

Suppression of c-myc expression and c-Myc function in response to sustained DNA damage in MCF-7 breast tumor cells.

The topoisomerase II inhibitors teniposide (VM-26), doxorubicin, and amsacrine (m-AMSA), as well as ionizing radiation, induce a transient suppression of c-myc mRNA, which correlates with growth inhibition of MCF-7 breast tumor cells. To further assess the involvement of c-mvc in the DNA damage-induced signal transduction pathways of the breast tumor cell, we determined the influence of sustained DNA damage on c-myc expression, c-Myc protein levels and c-Myc function. Continuous exposure of MCF-7 breast tumor cells to VM-26 induced DNA strand breaks that were sustained for at least 9 hr. DNA strand breakage was accompanied by a decline in c-myc transcripts and c-Myc protein levels by >90% after VM-26 exposure for 24 hr. The activity of a transcriptional target of the c-Myc protein, ornithine decarboxylase, was reduced by approximately 75% within 9 hr of DNA damage, in parallel to the declines in c-myc mRNA and protein levels. Extended exposure to VM-26 resulted in an initial loss of approximately 35% of the cell population followed by the death of additional cells such that by 72 hr only 50% of the cells were viable. Although apoptosis was evident 72 hr after initiating drug exposure [based on cell cycle analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, and an assessment of cell morphology], the primary phase of cell killing, which occurred during the first 24 hr was non-apoptotic. These studies indicate that non-apoptotic pathways can also mediate cell death in the breast tumor cell and support the role of c-myc expression, c-Myc protein, and c-Myc function as elements of the DNA damage response pathway in the breast tumor cell.

Effects of c-erbB2 overexpression on the drug sensitivities of normal human mammary epithelial cells.

BACKGROUND: Overexpression of the gene c-erbB2, which encodes a receptor tyrosine kinase, in breast tumors has been linked with either increased or decreased response of breast cancer patients to various therapies. In breast cancer cell lines, overexpression of exogenous c-erbB2 sometimes alters drug sensitivities but sometimes has no effect. To avoid the genetic complexities associated with established cancer cell lines, normal human mammary epithelial cells (HMECs) were studied to determine whether c-erbB2 overexpression by itself would alter chemosensitivity. METHODS: HMECs were designed to overexpress c-erbB2, and these cells were then evaluated for alterations in chemosensitivity. RESULTS: HMECs overexpressing c-erbB2 failed to show any alterations in chemosensitivity to a panel of chemotherapeutic agents, as indicated by 95% confidence intervals on growth curves of cells treated with or without the agent of interest. With the use of fluorescence-activated cell sorting to enrich for HMECs overexpressing c-erbB2 on their surface, an 85% pure population of cells was isolated and their chemosensitivity was evaluated. Again, the cells failed to display any alterations in chemosensitivity. CONCLUSIONS: These results suggest that overexpression of c-erbB2 is not sufficient by itself to induce changes in chemosensitivity. Cellular studies using normal human cells in which the complexity of the system can be carefully controlled by the addition of one, two, or even more genes associated with cancer development may provide valuable information about how the products of the genes interact with each other and which combinations are critical in regulating chemosensitivity.

UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase.

We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.

Induction of DNA damage, inhibition of DNA synthesis, and suppression of c-myc expression by the topoisomerase I inhibitor, camptothecin, in MCF-7 human breast tumor cells.

Previous work from this laboratory has demonstrated an association between the suppression of c-myc expression and the antiproliferative activity of both topoisomerase II inhibitors and ionizing radiation in MCF-7 breast tumor cells. These findings suggested that suppression of c-myc expression could be related to the induction of DNA damage in this cell line. The present studies were designed to determine whether the inhibition of topoisomerase I (and the consequent induction of DNA strand breaks) would also result in the suppression of c-myc expression. At camptothecin concentrations of 1 microM and below, there was no detectable damage (single- or double-strand breaks) in bulk DNA or suppression of c-myc expression. At camptothecin concentrations of 5, 10, and 25 microM, where suppression of c-myc expression was observed, strand breaks in bulk DNA were also detected. These findings are consistent with the idea that suppression of c-myc expression could be a component of the DNA damage response pathway in MCF-7 breast tumor cells. In contrast to the absence of detectable damage to bulk DNA or suppression of c-myc expression at the lower concentrations of camptothecin, DNA synthesis was inhibited over the entire range of drug concentrations and demonstrated a strong correspondence with growth inhibition. These observations support the concept that growth inhibition of MCF-7 cells by camptothecin is closely related to the early suppression of DNA synthesis.

Induction of DNA damage, inhibition of DNA synthesis and suppression of c-myc expression by the anthracycline analog, idarubicin (4-demethoxy-daunorubicin) in the MCF-7 breast tumor cell line.

PURPOSE: Studies were designed to elucidate the basis for the antiproliferative activity of the anthracycline antibiotic, idarubicin (4-demethoxy-daunorubicin) in MCF-7 breast tumor cells. METHODS: Growth inhibition was evaluated using the MTT tetrazolium dye assay, induction of DNA strand breaks was determined by alkaline elution, inhibition of DNA synthesis was assessed by measuring the incorporation of labelled thymidine into DNA, modulation of the expression of the c-myc oncogene was determined by Northern blotting and the induction of apoptosis was evaluated by alkaline unwinding, static field gel electrophoresis, terminal end labelling and assessment of cell morphology. RESULTS: MCF-7 cells were relatively sensitive to idarubicin, with an IC50 value for growth inhibition of approximately 0.01 microM. While DNA strand breakage was not evident below a concentration of 0.1 microM idarubicin, where growth inhibition exceeded 70%, both the inhibition of DNA synthesis and suppression of c-myc expression closely paralleled the profile of antiproliferative activity for idarubicin. Finally, while exposure to idarubicin resulted in a substantial loss of viable cells within 48-72 h, there was no morphological evidence of apoptotic body formation. The absence of apoptosis in cells exposed to idarubicin was supported by studies demonstrating the absence of DNA fragmentation using gel electrophoresis, alkaline elution and in situ DNA end-labelling assays. CONCLUSIONS: The results of these studies extend previous results from this laboratory indicating an association between suppression of c-myc expression, inhibition of DNA synthesis and growth arrest by topoisomerase II inhibitors, as well as the lack of induction of apoptotic cell death by topoisomerase II inhibitors in MCF-7 breast tumor cells.

An important role for the retinoblastoma protein in staurosporine-induced G1 arrest in murine embryonic fibroblasts.

In this study, we investigated the molecular basis of the ability of staurosporine to induce G1 arrest in murine embryonic fibroblasts (MEFs). We used MEFs from transgenic mice lacking several negative regulators of the G1/S phase transition including cells from mice lacking p53, p21, retinoblastoma (Rb), or p16 genes. We found that p53 function was not essential for staurosporine-induced G1 arrest. In contrast, MEFs from mice lacking Rb genes showed approximately a 70% reduced capacity to arrest in the G1 phase following staurosporine treatment. In support of a role for Rb in staurosporine-induced G1 arrest, rat embryonic fibroblasts stably overexpressing cyclin D1/Cdk4(R24C) exhibited approximately a 50% reduced G1 arrest response to staurosporine. The role of Rb in determining the degree of staurosporine-induced G1 arrest did not depend on the function of the cyclin-dependent kinase inhibitors p16 or p21 because MEFs lacking either of these genes were still capable of undergoing G1 arrest following staurosporine exposure. Our studies provide evidence of an important role for the Rb protein in determining the degree of staurosporine-induced G1 arrest in the first cell cycle.

Influence of ionizing radiation on proliferation, c-myc expression and the induction of apoptotic cell death in two breast tumour cell lines differing in p53 status.

PURPOSE: To determine the capacity of ionizing radiation to inhibit proliferation, to suppress c-myc expression and to induce apoptotic cell death in the p53 wild-type MCF-7 cell line and the p53 mutated MDA-MB231 cell line. MATERIALS AND METHODS: Growth inhibition and cell killing were determined by cell number and trypan blue exclusion. Apoptosis was assessed through cell morphology and fluorescent end-labelling. c-myc expression was monitored by Northern blotting. RESULTS: Inhibition of cell proliferation by ionizing radiation was similar in both cell lines. MDA-MB231 cells accumulated in G2 while MCF-7 cells accumulated in both the G1 and G2 phases of the cell cycle after irradiation. There was no evidence of apoptosis in either cell line. In MCF-7 cells, growth inhibition correlated closely with an early dose-dependent suppression of c-myc expression; in MDA-MB231 cells, there was no correspondence between growth inhibition and a transient, dose-independent reduction in c-myc message. CONCLUSIONS: These findings suggest that in the absence of classical apoptotic cell death, radiosensitivity is not predictably related to the p53 status of the cell. While both p53 and c-myc may be linked to the DNA damage response pathway, neither p53 nor c-myc are essential for growth arrest in response to ionizing radiation.

Ionizing radiation and teniposide increase p21(waf1/cip1) and promote Rb dephosphorylation but fail to suppress E2F activity in MCF-7 breast tumor cells.

Ionizing radiation and the topoisomerase II inhibitor, teniposide (VM-26) both increase levels of the cyclin dependent kinase inhibitor, p21(waf1/cip1) and promote dephosphorylation of the retinoblastoma tumor suppressor protein, Rb, in MCF-7 breast tumor cells, perturbations associated with suppression of the activity of the transcription factor, E2F. However, studies using an E2F binding site-luciferase reporter plasmid transfected into MCF-7 cells failed to demonstrate a reduction in E2F activity in response to VM-26 or to ionizing radiation. In contrast, E2F activity (both basal and E1A stimulated) could be suppressed by transfection with a plasmid expressing Rb, indicating that the capacity of E2F to bind to Rb and to be inactivated by Rb is functionally intact in MCF-7 cells. These findings in MCF-7 breast tumor cells suggest that E2F activity may not be directly susceptible to modulation by endogenous p21(waf1/cip1) and Rb.

Growth arrest and non-apoptotic cell death associated with the suppression of c-myc expression in MCF-7 breast tumor cells following acute exposure to doxorubicin.

In the MCF-7 human breast [correction of beast] adenocarcinoma cell line, acute exposure to 1 muM doxorubicin inhibited cell proliferation by approximately 75%. Analysis of cell cycle distribution indicated that within 24 hr, the G(2)/M fraction increased more than 3-fold and the S-phase population declined by >50%. In addition to growth arrest, there was an approximately 40% reduction in the viable cell population after 72 hr. Gel electrophoretic resolution of low molecular weight DNA immediately after exposure of cells to doxorubicin failed to demonstrate "laddered" oligonucleosomal profiles associated with apoptosis. The absence of intracellular DNA fragments or release of fragmented DNA into the incubation medium was confirmed by spectrofluorophotometry over a 72 hr interval following exposure of cells to 1 muM doxorubicin. In addition, there was no evidence of the morphological features associated with apoptosis during this period. Acute exposure to 1 muM doxorubicin also produced a transient increase in c-myc message expression (within the first hour) followed by a decline to 70% of control levels within 2-4 hr. The reduction in c-myc mRNA levels was concentration dependent and corresponded closely with growth arrest (as well as with inhibition of DNA synthesis). These findings (as well as similar reports demonstrating a correspondence between reduced c-myc expression and growth inhibition by VM-26 and m-AMSA in MCF-7 cells) suggest that the down-regulation of c-myc expression may reflect perturbations in regulatory processes contributing to growth arrest in MCF-7 cells exposed to topoisomerase II inhibitors.

Radiosensitization of HL-60 human leukaemia cells by bryostatin-1 in the absence of increased DNA fragmentation or apoptotic cell death.

Ionizing radiation produced a dose-dependent reduction in the proliferative capacity of HL-60 human promyelocytic leukaemia cells. A small percentage of the cell population demonstrated morphological evidence of apoptosis at 24h following radiation doses of > or = 5 Gy (i.e. 8% at 5 Gy and 16% at 10 Gy respectively) and produced a laddered oligonucleosomal pattern of DNA fragments by static-field gel electrophoresis. The antiproliferative effects of 1 and 2.5 Gy ionizing radiation were significantly enhanced by preincubating cells with bryostatin-1 at a concentration (10 nM) and time frame (24h) associated with down-regulation of total cellular protein kinase C (PKC) activity. Potentiation by bryostatin-1 of the radiation effect on proliferation was not associated with a concomitant increase in internucleosomal DNA fragmentation, in the fraction of cells exhibiting apoptotic morphology, or in the extent of radiation-induced single- or double-strand breaks in bulk DNA. Staurosporine, a potent but nonspecific inhibitor of PKC, was ineffective in altering the radiosensitivity of HL-60 cells or the degree of DNA fragmentation induced by ionizing radiation. These findings indicate that bryostatin 1 increases the sensitivity of human myeloid leukaemic cells to low radiation doses without enhancing DNA fragmentation or apoptosis, and that this capacity may involve factors other than, or in addition to, down-modulation of PKC activity.

Transcriptional down-regulation of c-myc expression in the MCF-7 breast tumor cell line by the topoisomerase II inhibitor, VM-26.

In the MCF-7 human breast tumor cell line, the topoisomerase II inhibitor, VM-26, produces a concentration dependent reduction in expression of the oncogene c-myc which parallels growth inhibition. Down-regulation of c-myc expression was examined at transcriptional and post-transcriptional levels. VM-26, at 10 microM, produced a reduction in the transcription rate of both sense and antisense strands of c-myc as determined by nuclear run-off analysis. In contrast, in the presence of the RNA synthesis inhibitor, actinomycin D, VM-26 failed to alter the half-life of the c-myc message. The capacity of VM-26 to reduce c-myc expression was not abrogated in cells pretreated with the protein synthesis inhibitor, cycloheximide (despite superinduction of c-myc expression in both control and VM-26 treated cells); this observation suggests that de novo protein synthesis may not be required to mediate the effects of VM-26 on steady state c-myc transcript levels. An extended analysis of the time course of c-myc expression demonstrated that the decline of steady state c-myc mRNA levels induced by VM-26 was biphasic, 6 h after the initial reduction in c-myc expression to approx. 30% of control levels, c-myc levels rebounded to 70% of control; after 24 h, c-myc expression declined gradually and remained at depressed levels (40% of control) at 48 and 72 h. These observations suggest that the initial transient reduction in c-myc expression associated with inhibition of transcription may represent a component of an early signalling pathway leading to growth arrest in MCF-7 breast tumor cells exposed to VM-26.

Induction of differentiation and growth arrest associated with nascent (nonoligosomal) DNA fragmentation and reduced c-myc expression in MCF-7 human breast tumor cells after continuous exposure to a sublethal concentration of doxorubicin.

The effects on DNA integrity of continuous (72-h) exposure of human MCF-7 breast adenocarcinoma cells to 50 nM doxorubicin (a concentration which can be maintained in the plasma by continuous infusion) were characterized by bisbenzimide spectrofluorophotometry, cell flow cytometry, agarose gel electrophoresis, and neutral elution. Spectrofluorophotometry and cell flow cytometry indicated the presence of DNA fragmentation, which was maximal at 24 h. Resolution of these fragments on agarose gels failed to demonstrate "laddered" oligosomal profiles. Neutral elution analysis at 24 h indicated that doxorubicin induced fragmentation of nascent, but not mature, double-stranded DNA. Drug-treated cells exhibited endoreduplication and significant shifts in cell cycle distribution, (i.e., increased G0/G1 and G2/M fractions and a markedly reduced S-phase fraction). These alterations occurred without inhibiting the incorporation of [3H]dThd into cellular DNA; in fact, both the rate and magnitude of [3H]dThd incorporation increased progressively. Doxorubicin also produced a sustained decline in c-myc mRNA levels that paralleled both growth arrest and induction of DNA fragmentation. Ultrastructural examination revealed morphological alterations consistent with the induction of differentiation (e.g., increased lipid content and mitochondrial density, appearance of tight junctions, and secretory ducts) and further suggested the possibility of autocatalysis (e.g., lipofuschin-containing vacuoles). A gradual decline in cell number was observed, with loss of approximately 35% of the cell population after 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of amsacrine (m-AMSA) on bulk and gene-specific DNA damage and c-myc expression in MCF-7 breast tumor cells.

In the MCF-7 human breast tumor cell line, the aminoacridine, m-AMSA, induces protein-associated DNA strand breaks consistent with inhibition of topoisomerase II. However, neither single-strand nor double-strand breaks in DNA, determined using conventional assays, show a consistent relationship with m-AMSA-induced inhibition of growth. In contrast, when DNA strand breaks are determined by alkaline unwinding under the high salt conditions of the alkaline unwinding/Southern blotting (AU/SB) assay, developed by our laboratories, damage to DNA corresponds closely with growth inhibition. The AU/SB assay, which is capable of assessing breaks within large-scale domains (upwards of 1 megabase) surrounding genes of interest, was further utilized to explore the capacity of m-AMSA to induce damage within specific genomic regions that may regulate cell growth. Regions encompassing the transcriptionally active oncogenes, c-myc and c-fos, were found to be more susceptible to m-AMSA-induced strand breaks than the region encompassing the non-transcribed alpha-satellite DNA or the genome as a whole (bulk DNA). These findings demonstrate that m-AMSA may produce more pronounced damage within specific genomic regions than in bulk DNA, m-AMSA also preferentially altered expression of the c-myc oncogene; at an m-AMSA concentration where growth was inhibited by between 70 and 80%, steady-state c-myc mRNA levels declined to approximately 10-15% of control levels within 2-3 hr; furthermore, concentration-dependent reductions in c-myc expression appeared to coincide with growth inhibition. In addition, inhibition of [3H]thymidine incorporation after 2 hr directly paralleled inhibition of growth, suggesting an early effect at the level of DNA biosynthesis, possibly related to the down-regulation of c-myc expression. It is proposed that specific lesions, e.g., in regions surrounding the c-myc gene, as well as generalized lesions in DNA may lead to growth inhibition mediated by down-regulation of the expression of select growth regulatory genes, such as c-myc.

Dissociation between bulk damage to DNA and the antiproliferative activity of teniposide (VM-26) in the MCF-7 breast tumor cell line: evidence for induction of gene-specific damage and alterations in gene expression.

In the MCF-7 breast tumor cell line, induction of bulk damage to DNA (measured either as total strand breaks or as double-strand breaks) fails to correspond with the antiproliferative activity of the demethylepipodo-phyllotoxin derivative, VM-26. In contrast, VM-26 produces an early (within 2-3 h) concentration-dependent reduction in c-myc expression (and of DNA synthesis) which parallels inhibition of cell growth, suggesting the possibility of effects of VM-26 at the level of genomic regions which regulate DNA replicative function. Although VM-26 also produces a reduction in c-myc expression in K562 human leukemic cells, these alterations fail to correspond with the concentration-dependent effects on cell growth in this cell line. Utilizing the newly developed alkaline unwinding/Southern blotting assay in the MCF-7 breast tumor cell line, it was determined that VM-26 induces damage within regions surrounding the c-myc gene and the beta-globin gene which exceeds that induced in both alpha-satellite DNA and in L1 repeat sequences; damage within c-myc and beta-globin also exceeds that observed throughout the genome as a whole. These findings indicate that certain genomic regions incur preferential damage in MCF-7 cells exposed to VM-26. It appears possible that damage within such genomic regions could lead to alterations in expression of select genes associated with regulation of cellular proliferation, resulting in reduced DNA synthesis, compromised cell growth, and, ultimately, cell death.