Francis Ali-Osman - Academia.edu (original) (raw)

Papers by Francis Ali-Osman

Research paper thumbnail of the ATR-Chk1 Pathway Independently of the Mismatch Repair Pathway

The presence of DNA damage initiates signaling through the ataxia-telangiectasia mutated kinase (... more The presence of DNA damage initiates signaling through the ataxia-telangiectasia mutated kinase (ATM) and the ATM-and the Rad3-related kinase (ATR), which phosphorylate, thus activating, the checkpoint kinases (Chk) 1 and 2, which leads to cell cycle arrest. The bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is cytotoxic primarily by inducing DNA monoadducts and ultimately, interstrand crosslinks, which block DNA replication. In this study, we investigated the activation of the ATR-Chk1 pathway in response to BCNU treatment and the dependency of this response on the DNA mismatch repair (MMR) capacity. Medulloblastoma cells were exposed to low and moderate doses of BCNU and the effects on this DNA damage signaling pathway examined. In response to BCNU, Chk1 was found to be phosphorylated at serine 345 and exhibited increased kinase activity. Caffeine and wortmannin, which are broad-spectrum inhibitors of ATM and ATR, reduced this phosphorylation. Cell cycle analysis further revealed an accumulation of cells in the S phase in response to BCNU, an effect that was attenuated by caffeine. Small interfering RNA knockdown of ATR also reduced Chk1 phosphorylation following exposure to BCNU. However, knockdown of ATM had no effect on the observed Chk1 phosphorylation, suggesting that ATR was primarily responsible for Chk1 activation. Analysis of Chk1 activation in cells deficient in MMR proteins MutLα or MutSα indicated that the DNA damage response induced by BCNU was independent of the MMR apparatus. This MMR-independent activation appears to be the result of DNA interstrand crosslink formation.

Research paper thumbnail of A Phase 2 Trial of Imatinib Mesylate As Maintenance Therapy for Patients with Newly Diagnosed C-Kit Positive Acute Myeloid Leukemia (AML)

Blood, 2012

3597 The c-kit (CD117) receptor is expressed on > 10% blasts in 64% of de novo AMLs and mediat... more 3597 The c-kit (CD117) receptor is expressed on > 10% blasts in 64% of de novo AMLs and mediates proliferation and anti-apoptotic effects. High c-kit levels correlate with a shorter time to relapse and decreased overall survival (OS). Imatinib mesylate (IM), a c-kit inhibitor, has activity against relapsed/refractory AML. The primary objective of this study was to determine whether adding maintenance IM for 1 yr after completion of standard induction (IT) and post-remission therapy (PRT) in patients (pts) with newly diagnosed c-kit+ AML improves relapse-free survival (RFS) compared to historical controls. Secondary objectives included: (1) assessing the feasibility of this approach; (2) evaluating outcomes based on c-kit expression (c-kit mean fluorescent intensity [MFI]); (3) determining whether c-kit expression correlates with AF1q gene and/or multi-drug resistance (MDR) gene expression. Methods: Pts were treated at Cleveland Clinic, Duke, Roswell Park, and University Hospitals...

Research paper thumbnail of EPID-12. Demographics and Lifestyle Factors in Glioma Risk: A report from the Glioma International Case-Control Study

Research paper thumbnail of Vergleich der Zytostatika-Empfindlichkeiten von L 1210-Zellen und menschlicher, stimulierter Lymphozyten in drei Zellproliferations-Assays

Research paper thumbnail of Culture of human normal brain and malignant brain tumors for cellular, molecular, and pharmacological studies

Methods in molecular medicine, 1996

Human brain neoplasms comprise a highly heterogeneous and biologically diverse group of tumors, t... more Human brain neoplasms comprise a highly heterogeneous and biologically diverse group of tumors, the most common and most malignant of which are those of neuroepithelial origin (1) Despite intensive research, little is still understood about the cellular and molecular processes involved in the genesis, progression, and response to therapy of these tumors. Much of the progress made to date, however, has resulted, in part from advances in the ability to culture and propagate cells of both normal and neoplastic brain tissue in vitro (2, 3) For example, in vitro cultures have contributed significantly to the development of techniques, such as bromodeoxyuridine labeling, that are used to estimate the cell-growth kinetics of gliomas in patients (4) Normal brain and brain tumor cultures have also played a central role in research directed at a better understanding of the complex interplay between the cellular components of the brain, such as that between various glial cells, neurons, and en...

Research paper thumbnail of Deletions and rearrangements inactivate the p16INK4 gene in human glioma cells

Oncogene, Jan 2, 1996

Structural alterations in the p16INK4 gene were examined in early passage human glioma cell lines... more Structural alterations in the p16INK4 gene were examined in early passage human glioma cell lines and related to the expression of p16 transcripts and protein. Using the Southern blot approach, we observed both homozygous and hemizygous deletions, as well as rearrangements of the p16 and p15 genes in 5 of the 7 cell lines (71%). Two cell lines, MGR3 and HBT28, revealed hemizygous deletion of the p16 and p15 genes combined with indistinguishable rearrangements of the remaining p15-p16 locus that resulted in loss of exon 2 sequences for p15 and p16, but retention of p16 exon 1; neither of these cell lines expressed p16 mRNA. Data for a third cell line, MGR2, indicated a similar, but unique rearrangement involving the p15 and p16 genes. MGR2, which retained a single wild-type p15-p16 locus, showed expression of p16 transcript, but not of p16 protein as indicated by Western blot analysis. All the glioma cell lines expressed similar levels of the retinoblastoma protein and no amplificati...

Research paper thumbnail of Formation and repair of 1,3-bis-(2-chloroethyl)-1-nitrosourea and cisplatin induced total genomic DNA interstrand crosslinks in human glioma cells

Cancer biochemistry biophysics, 1995

The kinetics of formation and repair of total genomic DNA interstrand crosslinks (ISCs) induced b... more The kinetics of formation and repair of total genomic DNA interstrand crosslinks (ISCs) induced by BCNU and cis-DDP were studied in cells of 6 human malignant gliomas and related with their degree of drug resistance. DNA ISCs were formed rapidly (peak 6-12 h) following a 2 h exposure to 50 microM BCNU or 25 uM cis-DDP, and on an equimolar basis higher levels of crosslinking were observed with cis-DDP than with BCNU. Repair of cis-DDP induced crosslinks was characteristically bi-phasic and the rate was significantly higher than that for BCNU induced crosslinks. Overall, a low crosslink index and a high crosslink repair rate correlated with cis-DDP and BCNU resistance. The data demonstrate, conclusively, the ability of human glioma cells to repair cis-DDP and, for the first time, BCNU induced DNA ISCs and that DNA crosslink repair is a significant contributing factor to the resistance of these tumors to the two agents.

Research paper thumbnail of Cytoprotection by somatostatin of normal and malignant clonogenic cells against the in vitro cytotoxicity of bischloroethylnitrosourea (BCNU)

Arzneimittel-Forschung, 1984

The various pharmacological effects of somatostatin may be explained by the hypothesis that the p... more The various pharmacological effects of somatostatin may be explained by the hypothesis that the paracrine peptide, by "stabilizing" cell membranes, inhibits the secretion of hormones as well as protects other cells (vascular endothelium, parenchyma) from different lesions (vasculo-, organo-, cytoprotection). This hypothesis was tested in vitro, using bischloroethyl-nitrosourea (BCNU)-intoxicated stem cells of normal mouse granulopoiesis and of the L 1210 leukemia. Clonogenic mouse bone marrow and L 1210 cells were grown in agar-containing glass capillaries. Using these colony assays and a ID90 of BCNU, cyclic somatostatin influenced the BCNU-cytotoxicity neither at simultaneous nor at subsequent application. However, when given 2 h prior to BCNU, the inhibition of colony growth was almost totally abolished. This cytoprotective effect was seen with normal granulopoietic as well as with leukemic cells. The effect did not show up, if the inactive linear somatostatin was used....

Research paper thumbnail of Mitogen-activated Protein Kinase (MAPK) Hyperactivation and Enhanced NRAS Expression Drive Acquired Vemurafenib Resistance in V600E BRAF Melanoma Cells

Journal of Biological Chemistry, 2014

Background: The response to vemurafenib in V600E BRAFϩve melanoma is short lived due to acquisiti... more Background: The response to vemurafenib in V600E BRAFϩve melanoma is short lived due to acquisition of vemurafenib resistance. Results: NRAS expression and increased MAPK activation drive vemurafenib resistance in V600E BRAFϩve melanoma. Conclusion: Resistance to vemurafenib in melanoma is complex and can be mitigated by MAPK and NRAS inhibition. Significance: These findings could lead to improved therapy of V600E BRAFϩve melanoma by targeting MAPKs and NRAS. Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF؉ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF؉ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF؉ve melanoma.

Research paper thumbnail of Rapid method for permanent slide preparation of colonies in soft agar cultures

The International Journal of Cell Cloning, 1986

A method is presented for preparing permanent microscopic slides from colony-bearing agar layers ... more A method is presented for preparing permanent microscopic slides from colony-bearing agar layers in soft agar cultures. The main advantages of this technique are its simplicity, rapidity and accurate colony preservation. This method could have broad applications in the human tumor clonogenic assay (HTCA), particularly in the quantitative morphological, cytochemical and immunocytochemical assessment of colonies that form in both control and drug-treated cultures. Thus, this method opens up possibilities for using cytopathological criteria as a quantitative endpoint of the HTCA.

Research paper thumbnail of Correlation of intralesional in vivo chemotherapy of line 10 hepatoma with in vitro drug sensitivity

The International Journal of Cell Cloning, 1983

The effects of intralesional chemotherapy with 7 different drugs on line 10 hepatoma grown in str... more The effects of intralesional chemotherapy with 7 different drugs on line 10 hepatoma grown in strain 2 guinea pigs were compared with the sensitivity of line 10 tumor cells in vitro, using a micro modification of the tumor stem cell assay with capillary tubes. A modified method was used to evaluate the in vitro dose-response curves. The correlation for in vivo/in vitro resistance was found to be 100% and for in vivo/in vitro sensitivity it was 80%.

Research paper thumbnail of Mechanism of the anti-tumour effect of biochemotherapy in melanoma: preliminary results

Research paper thumbnail of Primary Cultures of Human Glial Tumor Cells and Their Relationship to Histopathology

Journal of Neuropathology and Experimental Neurology, 1987

Research paper thumbnail of In vitro and in vivo investigations for the development of cytostatic methylhydrazones

Journal of Cancer Research and Clinical Oncology, 1985

In in vitro short-term (3 h) assays, the flchloroethyl-methyl-hydrazones B 1 and B 2 inhibit the ... more In in vitro short-term (3 h) assays, the flchloroethyl-methyl-hydrazones B 1 and B 2 inhibit the uptake of 3H-thymidine by EAC and L 1210 leukemia cells, B 2 being 5 to 10 times more effective than B 1. The growth inhibitory effect of both compounds was also confirmed in long-term (7 days) clonal assays using agar-containing glass capillaries, B 2 again being more effective than B 1. In contrast to these differences in vitro, in vivo both substances showed remission to the same degree in EAC-and complete resistance in L 1210-bearing mice. The diverging in vitro/in vivo sensitivities were thought to result from differences in the affinity of the methylhydrazones to the tumor cells: using short exposure periods (3 h) B 1 was more inhibitory than B 2 on both EAC and L 1210 colony growth; i.e., the more hydrophilic B 2 could more easily be washed off. To further test the idea of different cell membrane affinities, the methylhydrazones ZB 1 and P 1 with increasing lipophilic properties were synthesized. In vitro, after both pulse and continuous exposure ZB 1 and P 1 showed enforced inhibitory effects on colony growth. In vivo, ZB 1 and P 1 reduced the tumor weight of EAC mice, while only P 1 increased the survival time of L 1 210 mice. The results suggest that from the combination of in vitro/in vivo assays mechanistic conclusions can be derived that are valuable for further development of these cystostatics.

Research paper thumbnail of Comparison of cytostatic sensitivities of L 1210 cells and human stimulated lymphocytes in three cell proliferation assays

Journal of Cancer Research and Clinical Oncology, 1980

Three methods of measuring cell proliferation, viz., cellular 3H-thymidine uptake, counting of ce... more Three methods of measuring cell proliferation, viz., cellular 3H-thymidine uptake, counting of cells in suspension, and counting of colonies of cells grown in agar contained in glass capillaries, were compared by studying cell growth kinetics using the L 1210 cell line. We found the agar colony culture method to be most suitable and methodologically most advantageous. Using these cytokinetic models, we investigated the differential sensitivities of exponential and stationary phase L 1210 cells and normal, human, PHA-stimulated, peripheral T-lymphocytes to methotrexate, cytosine arabinoside, azathioprine, and a partially purified lymphocyte chalone preparation. L 1210 cells in exponential growth showed a higher drug sensitivity to all the agents tested than those in stationary growth. Normal, human T-lymphocytes exhibited less sensitivity to the tested agents. We found the agar culture to be more than twice as sensitive as the suspension culture and up to 8-fold more sensitive than the 3H-thymidine uptake method.

Research paper thumbnail of Translational inhibition of messenger RNA of the human π class glutathione S-transferase by antisense oligodeoxyribonucleotides

Chemico-Biological Interactions, 1998

In this study, a T7 plasmid expression vector containing the cDNA of a variant human GST-pi gene,... more In this study, a T7 plasmid expression vector containing the cDNA of a variant human GST-pi gene, hGSTP1*C, was used to examine the translational inhibition of the GST-pi mRNA with antisense deoxyribonucleotides (AS-ONs), and to investigate the dependency of the inhibition on ribonuclease (RNAse) H, AS-ON and target mRNA sequence specificity and AS-ON back bone modification. Translational inhibition of hGSTP1*C mRNA showed significant AS-ON concentration-dependency and was both target mRNA and AS-ON sequence specific. Fully modified phosphoromonthioate AS-ONs were less inhibitory than their partial phosphoromonthioate analogs; unmodified AS-ONs were inactive. RNAse H enhanced the translational inhibition by AS-ON specific to the translation initiation region mRNA, and was associated with cleavage of the target mRNA at the site of AS-ON:mRNA hybridization. AS-ONs directed to the A-->G and C-->T transitions, unique to hGSTP1*C, were more RNAse H-dependent than AS-ONs directed against the translation initiation site, indicating a greater involvement of RNAse H-dependent mRNA cleavage in the mechanism of translational inhibition by AS-ON at the polymorphic site. These data suggest that AS-ONs provide a potentially effective means of specific down-regulation of the human GST-pi gene, and demonstrate that the sites of GST-pi gene allelo-polymorphism can be targeted to translationally down-regulate the different GST-pi gene variants, specifically and differentially targeted.

Research paper thumbnail of Structure of the human allelic glutathione S-transferase-π gene variant, hGSTP1*C, cloned from a glioblastoma multiforme cell line

Chemico-Biological Interactions, 1998

We recently reported the cloning of full-length cDNAs corresponding to mRNAs of three GST-pi gene... more We recently reported the cloning of full-length cDNAs corresponding to mRNAs of three GST-pi genes, hGSTP1*A, hGSTP1*B and hGSTP1*C, as well as, the isolation of the full-length hGSTP1*C, of the human glutathione S-transferase-pi (GST-pi) gene that is characterized by a A-->G transition at +1404 in exon 5 and a C-->T transition at +2294 in exon 6. Although the promoter of the isolated gene was identical to that of the previously described GST-pi gene isolated from the MCF 7 and the HPB-ALL cell lines, both of which were hGSTP1*A, a number of structural differences were observed, including, nucleotide transitions, transversions, deletions and insertions, some of which created new restriction enzyme cleavage sites. A guanine insertion in the insulin response element, IRE, in intron 1 created an additional site for 5'-cytosine methylation. Seven repeat retinoic acid response element (RARE) consensus half sites, A(G)GG(T)TC(G)A at +1521 to +1644 were identified in the cloned hGSTP1*C. Five of the RARE half-sites had the minimal spacer nucleotide requirement for functionality and DNA mobility shift analysis with different pairs of the RARE half-sites and supershift studies using antibodies against RAR-beta showed significant binding of nuclear protein complexes from RA-treated cells to these RAREs. GST-pi gene expression was increased significantly in cells transfected with the GST-pi gene and treated with all-trans RA. These results contrast with those in a previous report in which RA was shown to suppress the GST-pi promoter, and indicate a complex mechanism of RA-mediated GST-pi gene regulation in tumor cells.

Research paper thumbnail of Abstract 2337: Defining significance of the novel tGLI1 transcription factor in cancer growth and progression

Cancer Research, 2011

The goal of this study is to gain a greater understanding of the molecular pathways that drive br... more The goal of this study is to gain a greater understanding of the molecular pathways that drive breast cancer progression and metastasis, the leading cause of breast cancer mortality. In achieving this goal, our research has been concentrated on GLI1 (glioma-associated oncogene homolog 1) zinc-finger transcription factor, the terminal effector of the Hedgehog pathway that is important for many physiological processes, constitutively activated in breast cancer and associated with poor survival of breast cancer patients. While no mutations in the human GLI1 gene have been reported in any cell or tumor type, we recently discovered a novel alternatively spliced truncated variant of GLI1, tGLI1, with an in-frame deletion of 41 codons spanning the entire exon 3 and part of exon 4 of the GLI1 gene (Can Res 17:6790-8, 2009). In the current study, we further discovered that the tGLI1 transcript and protein are highly expressed in the majority of cell lines and specimens of breast cancer we have examined to date, but is undetectable in normal cells or tissues. tGLI1 has retained the functional domains of GLI1 and a similar ability as GLI1 to undergo nuclear import and to activate GLI1-targeted genes. To gain insights into the role of tGLI1 in breast cancer progression, we used MDA-MB-231 cell line (known to be highly invasive and metastatic) to create three isogenic cell lines that stably express control vector, wild-type GLI1 and tGLI1. Functional characterization of these cell lines uncovered that tGLI1, but not wild-type GLI1, has rendered MDA-MB-231 cells significantly more invasive and migratory. This finding is also observed in other breast cancer cells we examined. Colony formation soft agar assays further indicate that tGLI1-expressing MDA-MB-231 cells had a higher propensity to undergo anchorage-independent growth than control cells and those with wild-type GLI1. To identify the molecular mechanisms that underlie tGLI1-associated phenotypes, we investigated the effects of tGLI1 (and wild-type GLI1) on several genes known to regulate cancer cell invasion, migration and growth. The results of these studies showed that tGLI1, but not GLI1, transcriptionally upregulates CD24 (an invasion/metastasis-associated gene) and downregulates PUMA (a proapoptotic gene) in breast cancer cells. In light of these exciting observations, ongoing efforts in our laboratory are to investigate the involvement of CD24 and PUMA (and other genes) in tGLI1-mediated breast cancer growth and progression. Animal studies are also in progress to examine the in vivo impact of tGLI1 on breast tumor invasion and metastasis. In summary, we report in this study that the newly discovered tGLI1 transcription factor is expressed tumor-specifically in breast cancer and associated with increased invasiveness, motility and anchorage-independent growth of breast cancer, and thus could be an important regulator of breast cancer growth and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2337. doi:10.1158/1538-7445.AM2011-2337

Research paper thumbnail of Repair analysis of 4-hydroperoxycyclophosphamide-induced DNA interstrand crosslinking in the c-myc gene in 4-hydroperoxycyclophosphamide-sensitive and-resistant medulloblastoma cell lines

Cancer Chemotherapy and Pharmacology, 1995

Cyclophosphamide is one of the most active agents in the treatment of medulloblastoma. However, d... more Cyclophosphamide is one of the most active agents in the treatment of medulloblastoma. However, development of resistance to this alkylator frequently occurs and is the harbinger of tumor progression and death. In order to understand the biochemical basis of this resistance, we generated a panel of medulloblastoma cell lines in our laboratory that were resistant to 4-hydroperoxycyclophosphamide (4-HC). Previously, we have shown that elevated levels of aldehyde dehydrogenase and glutathione mediate cellular resistance to 4-HC. The present study was conducted to identify the third unknown mechanism mediating the resistance of cell line D283 Med (4-HCR) to 4-HC, testing the hypothesis that this resistance is mediated by an increased repair of DNA interstrand crosslinks (ICLs). The doses of 4-HC that produced a one- and two-log cell kill of D283 Med cells were 25 and 50 microM, respectively, compared with values of 125 and 165 microM in D283 Med (4-HCR), the resistant cell line. The formation and disappearance of 4-HC-induced DNA ICLs at the c-myc gene were subsequently studied by DNA denaturing/renaturing gel electrophoresis and Southern blot analysis. 4-HC-induced DNA ICLs in the c-myc gene exhibited a dose-dependent relationship. The percentage of the c-myc gene that was crosslinked was approximately 1-3% at a dose of 100 microM. More than 50% of the DNA crosslinking in D283 Med (4-HCR) cells was removed by 6 h after drug treatment, whereas, in D283 Med cells, more than 90% of the DNA crosslinking was still present at 6 h. These findings suggest that the increased repair of DNA ICLs in D283 Med (4-HCR) may contribute significantly to its resistance to 4-HC.

Research paper thumbnail of Correlation of total and interstrand DNA adducts in tumor and kidney with antitumor efficacies and differential nephrotoxicities of cis-ammine/cyclohexylamine-dichloroplatinum(II) and cisplatin

Biochemical Pharmacology, 1994

Mixed amine platinum complexes have been identified as a new class of antitumor agents with activ... more Mixed amine platinum complexes have been identified as a new class of antitumor agents with activity in some cisplatin-resistant tumor models. cis-Ammine/cyclohexylamine-dichloroplatinum(II) is one such analog that we have evaluated in vivo and found it to have antitumor activity that was comparable to that of cisplatin in a solid murine fibrosarcoma tumor model. In contrast to the nephrotoxicity observed with cisplatin, the analog was free from inducing this side-effect. Pharmacokinetics of the two compounds administered i.v. at equitoxic dose levels to tumor-bearing mice indicated similar decay kinetics of total platinum in plasma, kidney and the tumor. Furthermore, DNA-platinum adducts of the two agents were similar in the tumor. Total adduct levels in the kidney, on the other hand, were significantly greater (P < 0.5) by up to 4-fold for cisplatin compared with the mixed amine analog. Likewise, the levels of interstrand cross-links of the two platinum complexes were comparable in the tumor, but significantly greater (P < 0.05) in the kidney for cisplatin. The data indicate that the greater renal levels of total and interstrand DNA-platinum adducts formed by cisplatin correlate with renal damage associated with this agent, and suggest that adduct levels, and not total tissue platinum levels, provide a more useful correlation with pharmacodynamic observations.

Research paper thumbnail of the ATR-Chk1 Pathway Independently of the Mismatch Repair Pathway

The presence of DNA damage initiates signaling through the ataxia-telangiectasia mutated kinase (... more The presence of DNA damage initiates signaling through the ataxia-telangiectasia mutated kinase (ATM) and the ATM-and the Rad3-related kinase (ATR), which phosphorylate, thus activating, the checkpoint kinases (Chk) 1 and 2, which leads to cell cycle arrest. The bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is cytotoxic primarily by inducing DNA monoadducts and ultimately, interstrand crosslinks, which block DNA replication. In this study, we investigated the activation of the ATR-Chk1 pathway in response to BCNU treatment and the dependency of this response on the DNA mismatch repair (MMR) capacity. Medulloblastoma cells were exposed to low and moderate doses of BCNU and the effects on this DNA damage signaling pathway examined. In response to BCNU, Chk1 was found to be phosphorylated at serine 345 and exhibited increased kinase activity. Caffeine and wortmannin, which are broad-spectrum inhibitors of ATM and ATR, reduced this phosphorylation. Cell cycle analysis further revealed an accumulation of cells in the S phase in response to BCNU, an effect that was attenuated by caffeine. Small interfering RNA knockdown of ATR also reduced Chk1 phosphorylation following exposure to BCNU. However, knockdown of ATM had no effect on the observed Chk1 phosphorylation, suggesting that ATR was primarily responsible for Chk1 activation. Analysis of Chk1 activation in cells deficient in MMR proteins MutLα or MutSα indicated that the DNA damage response induced by BCNU was independent of the MMR apparatus. This MMR-independent activation appears to be the result of DNA interstrand crosslink formation.

Research paper thumbnail of A Phase 2 Trial of Imatinib Mesylate As Maintenance Therapy for Patients with Newly Diagnosed C-Kit Positive Acute Myeloid Leukemia (AML)

Blood, 2012

3597 The c-kit (CD117) receptor is expressed on > 10% blasts in 64% of de novo AMLs and mediat... more 3597 The c-kit (CD117) receptor is expressed on > 10% blasts in 64% of de novo AMLs and mediates proliferation and anti-apoptotic effects. High c-kit levels correlate with a shorter time to relapse and decreased overall survival (OS). Imatinib mesylate (IM), a c-kit inhibitor, has activity against relapsed/refractory AML. The primary objective of this study was to determine whether adding maintenance IM for 1 yr after completion of standard induction (IT) and post-remission therapy (PRT) in patients (pts) with newly diagnosed c-kit+ AML improves relapse-free survival (RFS) compared to historical controls. Secondary objectives included: (1) assessing the feasibility of this approach; (2) evaluating outcomes based on c-kit expression (c-kit mean fluorescent intensity [MFI]); (3) determining whether c-kit expression correlates with AF1q gene and/or multi-drug resistance (MDR) gene expression. Methods: Pts were treated at Cleveland Clinic, Duke, Roswell Park, and University Hospitals...

Research paper thumbnail of EPID-12. Demographics and Lifestyle Factors in Glioma Risk: A report from the Glioma International Case-Control Study

Research paper thumbnail of Vergleich der Zytostatika-Empfindlichkeiten von L 1210-Zellen und menschlicher, stimulierter Lymphozyten in drei Zellproliferations-Assays

Research paper thumbnail of Culture of human normal brain and malignant brain tumors for cellular, molecular, and pharmacological studies

Methods in molecular medicine, 1996

Human brain neoplasms comprise a highly heterogeneous and biologically diverse group of tumors, t... more Human brain neoplasms comprise a highly heterogeneous and biologically diverse group of tumors, the most common and most malignant of which are those of neuroepithelial origin (1) Despite intensive research, little is still understood about the cellular and molecular processes involved in the genesis, progression, and response to therapy of these tumors. Much of the progress made to date, however, has resulted, in part from advances in the ability to culture and propagate cells of both normal and neoplastic brain tissue in vitro (2, 3) For example, in vitro cultures have contributed significantly to the development of techniques, such as bromodeoxyuridine labeling, that are used to estimate the cell-growth kinetics of gliomas in patients (4) Normal brain and brain tumor cultures have also played a central role in research directed at a better understanding of the complex interplay between the cellular components of the brain, such as that between various glial cells, neurons, and en...

Research paper thumbnail of Deletions and rearrangements inactivate the p16INK4 gene in human glioma cells

Oncogene, Jan 2, 1996

Structural alterations in the p16INK4 gene were examined in early passage human glioma cell lines... more Structural alterations in the p16INK4 gene were examined in early passage human glioma cell lines and related to the expression of p16 transcripts and protein. Using the Southern blot approach, we observed both homozygous and hemizygous deletions, as well as rearrangements of the p16 and p15 genes in 5 of the 7 cell lines (71%). Two cell lines, MGR3 and HBT28, revealed hemizygous deletion of the p16 and p15 genes combined with indistinguishable rearrangements of the remaining p15-p16 locus that resulted in loss of exon 2 sequences for p15 and p16, but retention of p16 exon 1; neither of these cell lines expressed p16 mRNA. Data for a third cell line, MGR2, indicated a similar, but unique rearrangement involving the p15 and p16 genes. MGR2, which retained a single wild-type p15-p16 locus, showed expression of p16 transcript, but not of p16 protein as indicated by Western blot analysis. All the glioma cell lines expressed similar levels of the retinoblastoma protein and no amplificati...

Research paper thumbnail of Formation and repair of 1,3-bis-(2-chloroethyl)-1-nitrosourea and cisplatin induced total genomic DNA interstrand crosslinks in human glioma cells

Cancer biochemistry biophysics, 1995

The kinetics of formation and repair of total genomic DNA interstrand crosslinks (ISCs) induced b... more The kinetics of formation and repair of total genomic DNA interstrand crosslinks (ISCs) induced by BCNU and cis-DDP were studied in cells of 6 human malignant gliomas and related with their degree of drug resistance. DNA ISCs were formed rapidly (peak 6-12 h) following a 2 h exposure to 50 microM BCNU or 25 uM cis-DDP, and on an equimolar basis higher levels of crosslinking were observed with cis-DDP than with BCNU. Repair of cis-DDP induced crosslinks was characteristically bi-phasic and the rate was significantly higher than that for BCNU induced crosslinks. Overall, a low crosslink index and a high crosslink repair rate correlated with cis-DDP and BCNU resistance. The data demonstrate, conclusively, the ability of human glioma cells to repair cis-DDP and, for the first time, BCNU induced DNA ISCs and that DNA crosslink repair is a significant contributing factor to the resistance of these tumors to the two agents.

Research paper thumbnail of Cytoprotection by somatostatin of normal and malignant clonogenic cells against the in vitro cytotoxicity of bischloroethylnitrosourea (BCNU)

Arzneimittel-Forschung, 1984

The various pharmacological effects of somatostatin may be explained by the hypothesis that the p... more The various pharmacological effects of somatostatin may be explained by the hypothesis that the paracrine peptide, by "stabilizing" cell membranes, inhibits the secretion of hormones as well as protects other cells (vascular endothelium, parenchyma) from different lesions (vasculo-, organo-, cytoprotection). This hypothesis was tested in vitro, using bischloroethyl-nitrosourea (BCNU)-intoxicated stem cells of normal mouse granulopoiesis and of the L 1210 leukemia. Clonogenic mouse bone marrow and L 1210 cells were grown in agar-containing glass capillaries. Using these colony assays and a ID90 of BCNU, cyclic somatostatin influenced the BCNU-cytotoxicity neither at simultaneous nor at subsequent application. However, when given 2 h prior to BCNU, the inhibition of colony growth was almost totally abolished. This cytoprotective effect was seen with normal granulopoietic as well as with leukemic cells. The effect did not show up, if the inactive linear somatostatin was used....

Research paper thumbnail of Mitogen-activated Protein Kinase (MAPK) Hyperactivation and Enhanced NRAS Expression Drive Acquired Vemurafenib Resistance in V600E BRAF Melanoma Cells

Journal of Biological Chemistry, 2014

Background: The response to vemurafenib in V600E BRAFϩve melanoma is short lived due to acquisiti... more Background: The response to vemurafenib in V600E BRAFϩve melanoma is short lived due to acquisition of vemurafenib resistance. Results: NRAS expression and increased MAPK activation drive vemurafenib resistance in V600E BRAFϩve melanoma. Conclusion: Resistance to vemurafenib in melanoma is complex and can be mitigated by MAPK and NRAS inhibition. Significance: These findings could lead to improved therapy of V600E BRAFϩve melanoma by targeting MAPKs and NRAS. Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF؉ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF؉ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF؉ve melanoma.

Research paper thumbnail of Rapid method for permanent slide preparation of colonies in soft agar cultures

The International Journal of Cell Cloning, 1986

A method is presented for preparing permanent microscopic slides from colony-bearing agar layers ... more A method is presented for preparing permanent microscopic slides from colony-bearing agar layers in soft agar cultures. The main advantages of this technique are its simplicity, rapidity and accurate colony preservation. This method could have broad applications in the human tumor clonogenic assay (HTCA), particularly in the quantitative morphological, cytochemical and immunocytochemical assessment of colonies that form in both control and drug-treated cultures. Thus, this method opens up possibilities for using cytopathological criteria as a quantitative endpoint of the HTCA.

Research paper thumbnail of Correlation of intralesional in vivo chemotherapy of line 10 hepatoma with in vitro drug sensitivity

The International Journal of Cell Cloning, 1983

The effects of intralesional chemotherapy with 7 different drugs on line 10 hepatoma grown in str... more The effects of intralesional chemotherapy with 7 different drugs on line 10 hepatoma grown in strain 2 guinea pigs were compared with the sensitivity of line 10 tumor cells in vitro, using a micro modification of the tumor stem cell assay with capillary tubes. A modified method was used to evaluate the in vitro dose-response curves. The correlation for in vivo/in vitro resistance was found to be 100% and for in vivo/in vitro sensitivity it was 80%.

Research paper thumbnail of Mechanism of the anti-tumour effect of biochemotherapy in melanoma: preliminary results

Research paper thumbnail of Primary Cultures of Human Glial Tumor Cells and Their Relationship to Histopathology

Journal of Neuropathology and Experimental Neurology, 1987

Research paper thumbnail of In vitro and in vivo investigations for the development of cytostatic methylhydrazones

Journal of Cancer Research and Clinical Oncology, 1985

In in vitro short-term (3 h) assays, the flchloroethyl-methyl-hydrazones B 1 and B 2 inhibit the ... more In in vitro short-term (3 h) assays, the flchloroethyl-methyl-hydrazones B 1 and B 2 inhibit the uptake of 3H-thymidine by EAC and L 1210 leukemia cells, B 2 being 5 to 10 times more effective than B 1. The growth inhibitory effect of both compounds was also confirmed in long-term (7 days) clonal assays using agar-containing glass capillaries, B 2 again being more effective than B 1. In contrast to these differences in vitro, in vivo both substances showed remission to the same degree in EAC-and complete resistance in L 1210-bearing mice. The diverging in vitro/in vivo sensitivities were thought to result from differences in the affinity of the methylhydrazones to the tumor cells: using short exposure periods (3 h) B 1 was more inhibitory than B 2 on both EAC and L 1210 colony growth; i.e., the more hydrophilic B 2 could more easily be washed off. To further test the idea of different cell membrane affinities, the methylhydrazones ZB 1 and P 1 with increasing lipophilic properties were synthesized. In vitro, after both pulse and continuous exposure ZB 1 and P 1 showed enforced inhibitory effects on colony growth. In vivo, ZB 1 and P 1 reduced the tumor weight of EAC mice, while only P 1 increased the survival time of L 1 210 mice. The results suggest that from the combination of in vitro/in vivo assays mechanistic conclusions can be derived that are valuable for further development of these cystostatics.

Research paper thumbnail of Comparison of cytostatic sensitivities of L 1210 cells and human stimulated lymphocytes in three cell proliferation assays

Journal of Cancer Research and Clinical Oncology, 1980

Three methods of measuring cell proliferation, viz., cellular 3H-thymidine uptake, counting of ce... more Three methods of measuring cell proliferation, viz., cellular 3H-thymidine uptake, counting of cells in suspension, and counting of colonies of cells grown in agar contained in glass capillaries, were compared by studying cell growth kinetics using the L 1210 cell line. We found the agar colony culture method to be most suitable and methodologically most advantageous. Using these cytokinetic models, we investigated the differential sensitivities of exponential and stationary phase L 1210 cells and normal, human, PHA-stimulated, peripheral T-lymphocytes to methotrexate, cytosine arabinoside, azathioprine, and a partially purified lymphocyte chalone preparation. L 1210 cells in exponential growth showed a higher drug sensitivity to all the agents tested than those in stationary growth. Normal, human T-lymphocytes exhibited less sensitivity to the tested agents. We found the agar culture to be more than twice as sensitive as the suspension culture and up to 8-fold more sensitive than the 3H-thymidine uptake method.

Research paper thumbnail of Translational inhibition of messenger RNA of the human π class glutathione S-transferase by antisense oligodeoxyribonucleotides

Chemico-Biological Interactions, 1998

In this study, a T7 plasmid expression vector containing the cDNA of a variant human GST-pi gene,... more In this study, a T7 plasmid expression vector containing the cDNA of a variant human GST-pi gene, hGSTP1*C, was used to examine the translational inhibition of the GST-pi mRNA with antisense deoxyribonucleotides (AS-ONs), and to investigate the dependency of the inhibition on ribonuclease (RNAse) H, AS-ON and target mRNA sequence specificity and AS-ON back bone modification. Translational inhibition of hGSTP1*C mRNA showed significant AS-ON concentration-dependency and was both target mRNA and AS-ON sequence specific. Fully modified phosphoromonthioate AS-ONs were less inhibitory than their partial phosphoromonthioate analogs; unmodified AS-ONs were inactive. RNAse H enhanced the translational inhibition by AS-ON specific to the translation initiation region mRNA, and was associated with cleavage of the target mRNA at the site of AS-ON:mRNA hybridization. AS-ONs directed to the A-->G and C-->T transitions, unique to hGSTP1*C, were more RNAse H-dependent than AS-ONs directed against the translation initiation site, indicating a greater involvement of RNAse H-dependent mRNA cleavage in the mechanism of translational inhibition by AS-ON at the polymorphic site. These data suggest that AS-ONs provide a potentially effective means of specific down-regulation of the human GST-pi gene, and demonstrate that the sites of GST-pi gene allelo-polymorphism can be targeted to translationally down-regulate the different GST-pi gene variants, specifically and differentially targeted.

Research paper thumbnail of Structure of the human allelic glutathione S-transferase-π gene variant, hGSTP1*C, cloned from a glioblastoma multiforme cell line

Chemico-Biological Interactions, 1998

We recently reported the cloning of full-length cDNAs corresponding to mRNAs of three GST-pi gene... more We recently reported the cloning of full-length cDNAs corresponding to mRNAs of three GST-pi genes, hGSTP1*A, hGSTP1*B and hGSTP1*C, as well as, the isolation of the full-length hGSTP1*C, of the human glutathione S-transferase-pi (GST-pi) gene that is characterized by a A-->G transition at +1404 in exon 5 and a C-->T transition at +2294 in exon 6. Although the promoter of the isolated gene was identical to that of the previously described GST-pi gene isolated from the MCF 7 and the HPB-ALL cell lines, both of which were hGSTP1*A, a number of structural differences were observed, including, nucleotide transitions, transversions, deletions and insertions, some of which created new restriction enzyme cleavage sites. A guanine insertion in the insulin response element, IRE, in intron 1 created an additional site for 5'-cytosine methylation. Seven repeat retinoic acid response element (RARE) consensus half sites, A(G)GG(T)TC(G)A at +1521 to +1644 were identified in the cloned hGSTP1*C. Five of the RARE half-sites had the minimal spacer nucleotide requirement for functionality and DNA mobility shift analysis with different pairs of the RARE half-sites and supershift studies using antibodies against RAR-beta showed significant binding of nuclear protein complexes from RA-treated cells to these RAREs. GST-pi gene expression was increased significantly in cells transfected with the GST-pi gene and treated with all-trans RA. These results contrast with those in a previous report in which RA was shown to suppress the GST-pi promoter, and indicate a complex mechanism of RA-mediated GST-pi gene regulation in tumor cells.

Research paper thumbnail of Abstract 2337: Defining significance of the novel tGLI1 transcription factor in cancer growth and progression

Cancer Research, 2011

The goal of this study is to gain a greater understanding of the molecular pathways that drive br... more The goal of this study is to gain a greater understanding of the molecular pathways that drive breast cancer progression and metastasis, the leading cause of breast cancer mortality. In achieving this goal, our research has been concentrated on GLI1 (glioma-associated oncogene homolog 1) zinc-finger transcription factor, the terminal effector of the Hedgehog pathway that is important for many physiological processes, constitutively activated in breast cancer and associated with poor survival of breast cancer patients. While no mutations in the human GLI1 gene have been reported in any cell or tumor type, we recently discovered a novel alternatively spliced truncated variant of GLI1, tGLI1, with an in-frame deletion of 41 codons spanning the entire exon 3 and part of exon 4 of the GLI1 gene (Can Res 17:6790-8, 2009). In the current study, we further discovered that the tGLI1 transcript and protein are highly expressed in the majority of cell lines and specimens of breast cancer we have examined to date, but is undetectable in normal cells or tissues. tGLI1 has retained the functional domains of GLI1 and a similar ability as GLI1 to undergo nuclear import and to activate GLI1-targeted genes. To gain insights into the role of tGLI1 in breast cancer progression, we used MDA-MB-231 cell line (known to be highly invasive and metastatic) to create three isogenic cell lines that stably express control vector, wild-type GLI1 and tGLI1. Functional characterization of these cell lines uncovered that tGLI1, but not wild-type GLI1, has rendered MDA-MB-231 cells significantly more invasive and migratory. This finding is also observed in other breast cancer cells we examined. Colony formation soft agar assays further indicate that tGLI1-expressing MDA-MB-231 cells had a higher propensity to undergo anchorage-independent growth than control cells and those with wild-type GLI1. To identify the molecular mechanisms that underlie tGLI1-associated phenotypes, we investigated the effects of tGLI1 (and wild-type GLI1) on several genes known to regulate cancer cell invasion, migration and growth. The results of these studies showed that tGLI1, but not GLI1, transcriptionally upregulates CD24 (an invasion/metastasis-associated gene) and downregulates PUMA (a proapoptotic gene) in breast cancer cells. In light of these exciting observations, ongoing efforts in our laboratory are to investigate the involvement of CD24 and PUMA (and other genes) in tGLI1-mediated breast cancer growth and progression. Animal studies are also in progress to examine the in vivo impact of tGLI1 on breast tumor invasion and metastasis. In summary, we report in this study that the newly discovered tGLI1 transcription factor is expressed tumor-specifically in breast cancer and associated with increased invasiveness, motility and anchorage-independent growth of breast cancer, and thus could be an important regulator of breast cancer growth and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2337. doi:10.1158/1538-7445.AM2011-2337

Research paper thumbnail of Repair analysis of 4-hydroperoxycyclophosphamide-induced DNA interstrand crosslinking in the c-myc gene in 4-hydroperoxycyclophosphamide-sensitive and-resistant medulloblastoma cell lines

Cancer Chemotherapy and Pharmacology, 1995

Cyclophosphamide is one of the most active agents in the treatment of medulloblastoma. However, d... more Cyclophosphamide is one of the most active agents in the treatment of medulloblastoma. However, development of resistance to this alkylator frequently occurs and is the harbinger of tumor progression and death. In order to understand the biochemical basis of this resistance, we generated a panel of medulloblastoma cell lines in our laboratory that were resistant to 4-hydroperoxycyclophosphamide (4-HC). Previously, we have shown that elevated levels of aldehyde dehydrogenase and glutathione mediate cellular resistance to 4-HC. The present study was conducted to identify the third unknown mechanism mediating the resistance of cell line D283 Med (4-HCR) to 4-HC, testing the hypothesis that this resistance is mediated by an increased repair of DNA interstrand crosslinks (ICLs). The doses of 4-HC that produced a one- and two-log cell kill of D283 Med cells were 25 and 50 microM, respectively, compared with values of 125 and 165 microM in D283 Med (4-HCR), the resistant cell line. The formation and disappearance of 4-HC-induced DNA ICLs at the c-myc gene were subsequently studied by DNA denaturing/renaturing gel electrophoresis and Southern blot analysis. 4-HC-induced DNA ICLs in the c-myc gene exhibited a dose-dependent relationship. The percentage of the c-myc gene that was crosslinked was approximately 1-3% at a dose of 100 microM. More than 50% of the DNA crosslinking in D283 Med (4-HCR) cells was removed by 6 h after drug treatment, whereas, in D283 Med cells, more than 90% of the DNA crosslinking was still present at 6 h. These findings suggest that the increased repair of DNA ICLs in D283 Med (4-HCR) may contribute significantly to its resistance to 4-HC.

Research paper thumbnail of Correlation of total and interstrand DNA adducts in tumor and kidney with antitumor efficacies and differential nephrotoxicities of cis-ammine/cyclohexylamine-dichloroplatinum(II) and cisplatin

Biochemical Pharmacology, 1994

Mixed amine platinum complexes have been identified as a new class of antitumor agents with activ... more Mixed amine platinum complexes have been identified as a new class of antitumor agents with activity in some cisplatin-resistant tumor models. cis-Ammine/cyclohexylamine-dichloroplatinum(II) is one such analog that we have evaluated in vivo and found it to have antitumor activity that was comparable to that of cisplatin in a solid murine fibrosarcoma tumor model. In contrast to the nephrotoxicity observed with cisplatin, the analog was free from inducing this side-effect. Pharmacokinetics of the two compounds administered i.v. at equitoxic dose levels to tumor-bearing mice indicated similar decay kinetics of total platinum in plasma, kidney and the tumor. Furthermore, DNA-platinum adducts of the two agents were similar in the tumor. Total adduct levels in the kidney, on the other hand, were significantly greater (P < 0.5) by up to 4-fold for cisplatin compared with the mixed amine analog. Likewise, the levels of interstrand cross-links of the two platinum complexes were comparable in the tumor, but significantly greater (P < 0.05) in the kidney for cisplatin. The data indicate that the greater renal levels of total and interstrand DNA-platinum adducts formed by cisplatin correlate with renal damage associated with this agent, and suggest that adduct levels, and not total tissue platinum levels, provide a more useful correlation with pharmacodynamic observations.