Kathy Vassilakos - Academia.edu (original) (raw)

Papers by Kathy Vassilakos

Research paper thumbnail of Preclinical toxicity and toxicokinetics of GTI-2040, a phosphorothioate oligonucleotide targeting ribonucleotide reductase R2

Cancer Chemotherapy and Pharmacology, 2010

Purpose GTI-2040, a 20-mer phosphorothioate oligonucleotide, was designed to hybridize to the mRN... more Purpose GTI-2040, a 20-mer phosphorothioate oligonucleotide, was designed to hybridize to the mRNA sequence of human ribonucleotide reductase R2. GTI-2040 has been shown to inhibit human cancer cell proliferation by downregulation of R2 expression in vitro and to signiWcantly inhibit tumor growth in xenograft models of human cancer in mice. As part of the safety evaluation for human clinical trials, the toxicity and toxicokinetics of GTI-2040 were determined in Sprague-Dawley rats and rhesus monkeys. Methods GTI-2040 was administered to rats at 2, 10, and 50 mg/kg/day by bolus intravenous injection every second day for 21 days with a 21-day recovery. In monkeys, an acute study was performed with single, escalating doses of GTI-2040 ranging from 10 to 80 mg/kg given as a 24-h continuous intravenous infusion. As well, a 21-day, continuous intravenous infusion study with GTI-2040 was conducted in monkeys at 2, 10, and 50 mg/kg/day, with a 3-week recovery. Blood sampling was done to measure GTI-2040 plasma concentrations, metabolites, and pharmacokinetic parameters, and tissues were collected to assess the distribution of GTI-2040 and/or metabolites. Results The toxicities of GTI-2040 in both rats and monkeys were typical for the phosphorothioate oligonucleotide class of compounds. In monkeys, there was a dose-related increase in GTI-2040 plasma levels with concomitant increase in complement activation and prolongation of activated partial thromboplastin time. In both rats and monkeys, the tissues having the highest concentrations of GTI-2040 (kidney, liver, spleen) had the largest doserelated toxic eVects. Adverse eVects were diminished or absent in the recovery animals. Conclusions GTI-2040 was well tolerated when infused over 24 h at doses up to 80 mg/kg in monkeys. In rats and monkeys, GTI-2040 was reasonably well tolerated and showed reversible toxicities when administered at doses up to 50 mg/kg/day for 21 days. The no observed adverse eVect dose level for GTI-2040 in both animal species was 2 mg/kg/day. There were no apparent sequence-speciWc eVects related to the interaction of GTI-2040 with the R2 component of the mRNA expressing ribonucleotide reductase.

Research paper thumbnail of Journal of Clinical Laboratory Analysis 19:182–188 (2005) Optimization of the PAXgene TM Blood RNA Extraction System for Gene Expression Analysis of Clinical Samples

One major problem associated with collecting whole blood from patients for use as a source of RNA... more One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene TM blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene TM to stabilize RNA in whole blood; however, RNA extracted by the PAXgene TM method co...

Research paper thumbnail of The molecular chaperones, calnexin and calreticulin, studies on function and mechanism of action

Research paper thumbnail of Studies on Insulin Receptor in Xenopus Laevis Oocytes

Mina Falcone fer her friendship, support and interest. I could always count on her technical advi... more Mina Falcone fer her friendship, support and interest. I could always count on her technical advice, but more importantly I could count on her honest opinion. Jason Young, Gil Abisdris, Josie Ursini and all the people who passed through the Andrews lab for many useful discussions, both scientific and otherwise, and especially for their friendship. Paul MacPherson for his patience, love and support. Without him I would be computer illiterate and often stranded at 3 a.m. without transportation home. My sister Helen, my niece Erin, my mother Panagiota and Jim and Mary MacPherson for their love and support over the years.

Research paper thumbnail of The 3 ' untranslated region of bovine preprolactin contains a transferable non-poly(A) mRNA sequence that prolongs

Research paper thumbnail of GTI-2040, an antisense agent targeting the small subunit component (R2) of human ribonucleotide reductase, shows potent antitumor activity against a variety of tumors

Cancer research, 2003

GTI-2040 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the ... more GTI-2040 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the R2 small subunit component of human ribonucleotide reductase. In vitro studies using a number of human tumor cell lines have demonstrated that GTI-2040 decreases mRNA and protein levels of R2 in a sequence- and target-specific manner. In vivo studies have shown that GTI-2040 significantly inhibits growth of human colon tumors (adenocarcinoma), pancreatic tumors (adenocarcinoma), liver tumors, lung tumors, breast tumors (adenocarcinoma), renal tumors, ovarian tumors (adenocarcinoma), melanoma, brain glioblastoma-astrocytoma, prostatic tumors, and cervical tumors in nude and/or severe combined immunodeficient mice. Antitumor effects were not observed with an oligonucleotide containing four mismatches to the R2 sequence or with a scrambled sequence containing the same base content but not complementary to R2. This suggests that an antisense mechanism is responsible for the in vivo observati...

Research paper thumbnail of Anti-proliferative and anti-tumor effects of antisense oligonucleotide GTI-2601 targeted against human thioredoxin

Anti Cancer Drugs, Mar 1, 2006

Human thioredoxin has been implicated in cancer as a growth stimulator through regulation of DNA ... more Human thioredoxin has been implicated in cancer as a growth stimulator through regulation of DNA replication and growth factor activity, as a modulator of transcription factor activity, and as an inhibitor of apoptosis. In the present study, the steady-state level of thioredoxin protein was examined in a number of cancer cell lines. Interestingly, thioredoxin expression is elevated in a variety of human tumor cell lines compared with normal cell lines. The altered expression of thioredoxin in tumor cells suggests it may be a target in the development of novel therapeutic agents for the treatment and prevention of cancer. Further to this possibility, 26 phosphorothioate antisense oligodeoxynucleotides (PS-AS-ODNs) were evaluated for the ability to inhibit thioredoxin expression in cell culture. One PS-AS-ODN, GTI-2601, specifically reduced the levels of thioredoxin mRNA and protein, exhibited potent anti-proliferative effects on colony formation in vitro, and had anti-tumor effects in human tumor xenograft mouse models in vivo. Sequence-specific decreases in thioredoxin expression levels were accompanied by significant suppression of tumor growth in mice. Taken together, these data suggest that thioredoxin may be a useful target for developing PS-AS-ODNs as drug candidates against human cancer.

Research paper thumbnail of Adenovirus-Mediated Ribonucleotide Reductase R1 Gene Therapy of Human Colon Adenocarcinoma

Clinical Cancer Research, Oct 1, 2003

Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their ... more Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides for DNA synthesis. Ribonucleotide reductase is a multisubunit complex containing two polypeptides, R1 and R2. In addition to catalytic and allosteric regulatory functions, the R1 subunit appears to act as a novel tumor suppressor. Previous studies demonstrated that overexpression of mouse R1 resulted in suppression of tumorigenicity and metastatic potential, whereas expression of antisense RNA, complementary to R1 mRNA, increased anchorage-independent growth of ras-transformed NIH 3T3 cells. The current study investigated the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1).

Research paper thumbnail of Oligosaccharide Binding Characteristics of the Molecular Chaperones Calnexin and Calreticulin †

Biochemistry Usa, Mar 10, 1998

Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their... more Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. We compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, we show that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibited the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. We also examined the effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin. Whereas oligosaccharide binding was only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogated oligosaccharide binding, an effect that was largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins.

Research paper thumbnail of Optimization of the PAXgene TM Blood RNA Extraction System for Gene Expression Analysis of Clinical Samples

J Clin Lab Anal, 2005

One major problem associated with collecting whole blood from patients for use as a source of RNA... more One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene TM blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene TM to stabilize RNA in whole blood; however, RNA extracted by the PAXgene TM method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene TM protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene TM is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Research paper thumbnail of Peptide presentation by MHC class I molecules

Trends in cell biology, 1996

The presentation of peptides by class I histocompatibility molecules plays a central role in the ... more The presentation of peptides by class I histocompatibility molecules plays a central role in the cellular immune response to virally infected or transformed cells. The main steps in this process include the degradation of both self and 'foreign' proteins to short peptides in the cytosol, translocation of peptides into the lumen of the endoplasmic reticulum, binding of a subset of peptides to assembling class I molecules and expression of class-I-peptide complexes at the cell surface for examination by cytotoxic T cells. A molecular understanding of most of these steps is emerging, revealing a remarkable coordination between the processes of peptide translocation, delivery and binding to class I molecules.

Research paper thumbnail of The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules

The EMBO journal, 1996

Calnexin, a membrane protein of the endoplasmic reticulum, is generally thought to function as a ... more Calnexin, a membrane protein of the endoplasmic reticulum, is generally thought to function as a molecular chaperone, based on indirect or correlative evidence. To examine calnexin's functions more directly, we reconstituted the assembly of class I histocompatibility molecules in the absence or presence of calnexin in Drosophila melanogaster cells. Calnexin enhanced the assembly of class I heavy chains with beta 2-microglobulin as much as 5-fold. The improved assembly appeared largely due to more efficient folding of heavy chains, as evidenced by increased reactivity with a conformation-sensitive monoclonal antibody and by a reduction in the level of aggregates. Similar findings were obtained in mouse or human cells when the interaction of calnexin with class I heavy chains was prevented by treatment with the oligosaccharide processing inhibitor castanospermine. The ability of calnexin to facilitate castanospermine. The ability of calnexin to facilitate heavy chain folding and t...

Research paper thumbnail of GTI-2501, an antisense agent targeting R1, the large subunit of human ribonucleotide reductase, shows potent anti-tumor activity against a variety of tumors

International journal of oncology, 2006

GTI-2501 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of R1, ... more GTI-2501 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of R1, the large subunit of ribonucleotide reductase (RNR). In vitro studies, have demonstrated that GTI-2501 decreases mRNA and protein levels of R1 in a sequence-specific and dose-dependent manner. Furthermore, GTI-2501 inhibits the growth of human lung, liver, ovary, brain, melanoma, breast and pancreatic tumor cells in colony forming assays. In vivo studies have shown that GTI-2501 significantly inhibits growth of human colon, pancreas, lung, breast, renal, ovarian, melanoma, brain glioblastoma-astrocytoma, and prostatic tumors in CD-1 nude, Balb/c nude and/or SCID mice. GTI-2501 treatment caused total regression of human breast and renal tumor xenografts in mice. These effects are not observed with a scrambled control oligonucleotide containing the same base content but not complementary to R1. GTI-2501 specifically inhibits metastasis of human melanoma cells to the lungs in CD-1 athymic n...

Research paper thumbnail of GTI-2040, an antisense agent targeting the small subunit component (R2) of human ribonucleotide reductase, shows potent antitumor activity against a variety of tumors

Cancer research, 2003

GTI-2040 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the ... more GTI-2040 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the R2 small subunit component of human ribonucleotide reductase. In vitro studies using a number of human tumor cell lines have demonstrated that GTI-2040 decreases mRNA and protein levels of R2 in a sequence- and target-specific manner. In vivo studies have shown that GTI-2040 significantly inhibits growth of human colon tumors (adenocarcinoma), pancreatic tumors (adenocarcinoma), liver tumors, lung tumors, breast tumors (adenocarcinoma), renal tumors, ovarian tumors (adenocarcinoma), melanoma, brain glioblastoma-astrocytoma, prostatic tumors, and cervical tumors in nude and/or severe combined immunodeficient mice. Antitumor effects were not observed with an oligonucleotide containing four mismatches to the R2 sequence or with a scrambled sequence containing the same base content but not complementary to R2. This suggests that an antisense mechanism is responsible for the in vivo observati...

Research paper thumbnail of Adenovirus-mediated ribonucleotide reductase R1 gene therapy of human colon adenocarcinoma

Clinical cancer research : an official journal of the American Association for Cancer Research, 2003

Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their ... more Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides for DNA synthesis. Ribonucleotide reductase is a multisubunit complex containing two polypeptides, R1 and R2. In addition to catalytic and allosteric regulatory functions, the R1 subunit appears to act as a novel tumor suppressor. Previous studies demonstrated that overexpression of mouse R1 resulted in suppression of tumorigenicity and metastatic potential, whereas expression of antisense RNA, complementary to R1 mRNA, increased anchorage-independent growth of ras-transformed NIH 3T3 cells. The current study investigated the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1). Recombinant viruses were constructed by FLP-mediated site-specific recombination and demonstrated high infectivity of a human colon carcinoma cell line (Colo320 HRS), as assessed by expression of a viral encoded beta-G...

Research paper thumbnail of GTI-2040 displays cooperative anti-tumor activity when combined with interferon α against human renal carcinoma xenografts

International Journal of Oncology, 1992

, an antisense oligonucleotide targeting the small subunit of ribonucleotide reductase, acts as a... more , an antisense oligonucleotide targeting the small subunit of ribonucleotide reductase, acts as an anti-tumor agent in animal models of human cancer. In the present study, the anti-tumor activity of GTI-2040, in combination with interferon · (IFN·) was investigated against human renal cell carcinoma tumors xenografted into mice. The human renal cell carcinoma cell lines, Caki-1 and A498 were sensitive to IFN· both in vitro and when implanted into mice. In combination with GTI-2040 there were cooperative effects at intermediate doses of the two agents and complete tumor regression at higher combination doses. A control oligonucleotide was not effective as a monotherapy and did not improve the efficacy of IFN·. The effect of combination treatment on apoptosis and proliferation of tumor cells, isolated from xenografted tumors, was examined by histochemistry. GTI-2040 increased the percentage of cells undergoing apoptosis with a concomitant decrease in proliferation. IFN· alone had no effect but in combination with GTI-2040 resulted in increased apoptosis and decreased proliferation compared to GTI-2040 alone. Taken together these results expand the potential clinical applications of GTI-2040 to include combination therapy with IFN·.

Research paper thumbnail of Analysis of ribonucleotide reductase M2 mRNA levels in patient samples after GTI-2040 antisense drug treatment

Oncology Reports, 2006

This study describes the development of a rapid and practical real-time RT-PCR method to quantify... more This study describes the development of a rapid and practical real-time RT-PCR method to quantify ribonucleotide reductase M2 (RRM2) mRNA in tumor and peripheral white blood cells (WBCs) from patients treated with GTI-2040, an antisense drug currently in clinical trials. In order to assess target down-regulation by GTI-2040, RRM2 mRNA expression levels were analyzed in pre-and post-treatment samples from a phase II clinical trial of GTI-2040 combined with capecitabine in patients with metastatic breast cancer. Target gene RRM2 mRNA levels were evaluated using quantitative RT-PCR method: real-time PCR (TaqMan ® ) with fluorescein labeled probes on an ABI 7900HT instrument, with additional post-processing of the data to adjust for differences in total RNA in-put across the samples. Data are presented from a patient for whom both biopsy and PBMC samples were available, demonstrating applicability of this reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of mRNAs for RRM2 in human WBC and tissue samples. By providing quantitative measurement of changes in target gene expression, this method may provide an opportunity to determine the correlation between target response to GTI-2040 antisense and clinical response in patients. Furthermore this assay may assess whether WBC samples are an appropriate surrogate tissue for approximating target down-regulation in the tumor.

Research paper thumbnail of NC381, a Novel Anticancer Agent, Arrests the Cell Cycle in G0-G1 and Inhibits Lung Tumor Cell Growth in Vitro and in Vivo

Journal of Pharmacology and Experimental Therapeutics, 2003

Although clotrimazole (CLT), an antifungal drug, inhibits tumor cell proliferation and angiogenes... more Although clotrimazole (CLT), an antifungal drug, inhibits tumor cell proliferation and angiogenesis, its clinical application is hampered by significant hepatotoxicity due to the presence of an imidazole moiety. In our attempts to develop CLT analogs that are devoid of imidazole and are as efficacious as CLT, one pharmacophore designated NC381 was generated and shown to inhibit tumor cell growth via a mechanism similar to that of CLT. In vitro, treatment of NCI-H460 nonsmall cell lung cancer (NSCLC) cells with NC381 inhibited growth in a time-dependent manner. Flow cytometric analysis demonstrated that the decrease in cell growth was associated with inhibition of cell cycle progression at the G(1)-S phase transition, resulting in G(0)-G(1) arrest. There was a concomitant inhibition of cyclin D1 expression and subsequent reduction in the formation of the cyclin D1-CDK4 complex. Consistent with a decrease in the cyclin D1-CDK4 complex, NC381 treatment resulted in significant inhibition of pRb phosphorylation. There also were changes in the activity of cell cycle-related proteins, including p16(Ink4) and p27(Kip1). Together, these results are consistent with a model in which NC381 arrests cell cycle progression via inhibition of the pathway that promotes exit from the G(1) phase of the cell cycle. Furthermore, the clinical applicability of NC381 was evaluated in an in vivo murine xenograft model of human NSCLC (NCI-H460). NC381 treatment resulted in significant inhibition of tumor growth. Given the poor prognosis and the limited treatment options available, the present results underscore the potential of NC381 in the treatment of human NSCLC.

Research paper thumbnail of Optimization of the PAXgene™ blood RNA extraction system for gene expression analysis of clinical samples

Journal of Clinical Laboratory Analysis, 2005

One major problem associated with collecting whole blood from patients for use as a source of RNA... more One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene TM blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene TM to stabilize RNA in whole blood; however, RNA extracted by the PAXgene TM method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene TM protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene TM is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Research paper thumbnail of The Molecular Chaperone Calnexin Binds Glc(1)Man(9)GlcNAc(2) Oligosaccharide as an Initial Step in Recognizing Unfolded Glycoproteins

Journal of Biological Chemistry, 1995

Research paper thumbnail of Preclinical toxicity and toxicokinetics of GTI-2040, a phosphorothioate oligonucleotide targeting ribonucleotide reductase R2

Cancer Chemotherapy and Pharmacology, 2010

Purpose GTI-2040, a 20-mer phosphorothioate oligonucleotide, was designed to hybridize to the mRN... more Purpose GTI-2040, a 20-mer phosphorothioate oligonucleotide, was designed to hybridize to the mRNA sequence of human ribonucleotide reductase R2. GTI-2040 has been shown to inhibit human cancer cell proliferation by downregulation of R2 expression in vitro and to signiWcantly inhibit tumor growth in xenograft models of human cancer in mice. As part of the safety evaluation for human clinical trials, the toxicity and toxicokinetics of GTI-2040 were determined in Sprague-Dawley rats and rhesus monkeys. Methods GTI-2040 was administered to rats at 2, 10, and 50 mg/kg/day by bolus intravenous injection every second day for 21 days with a 21-day recovery. In monkeys, an acute study was performed with single, escalating doses of GTI-2040 ranging from 10 to 80 mg/kg given as a 24-h continuous intravenous infusion. As well, a 21-day, continuous intravenous infusion study with GTI-2040 was conducted in monkeys at 2, 10, and 50 mg/kg/day, with a 3-week recovery. Blood sampling was done to measure GTI-2040 plasma concentrations, metabolites, and pharmacokinetic parameters, and tissues were collected to assess the distribution of GTI-2040 and/or metabolites. Results The toxicities of GTI-2040 in both rats and monkeys were typical for the phosphorothioate oligonucleotide class of compounds. In monkeys, there was a dose-related increase in GTI-2040 plasma levels with concomitant increase in complement activation and prolongation of activated partial thromboplastin time. In both rats and monkeys, the tissues having the highest concentrations of GTI-2040 (kidney, liver, spleen) had the largest doserelated toxic eVects. Adverse eVects were diminished or absent in the recovery animals. Conclusions GTI-2040 was well tolerated when infused over 24 h at doses up to 80 mg/kg in monkeys. In rats and monkeys, GTI-2040 was reasonably well tolerated and showed reversible toxicities when administered at doses up to 50 mg/kg/day for 21 days. The no observed adverse eVect dose level for GTI-2040 in both animal species was 2 mg/kg/day. There were no apparent sequence-speciWc eVects related to the interaction of GTI-2040 with the R2 component of the mRNA expressing ribonucleotide reductase.

Research paper thumbnail of Journal of Clinical Laboratory Analysis 19:182–188 (2005) Optimization of the PAXgene TM Blood RNA Extraction System for Gene Expression Analysis of Clinical Samples

One major problem associated with collecting whole blood from patients for use as a source of RNA... more One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene TM blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene TM to stabilize RNA in whole blood; however, RNA extracted by the PAXgene TM method co...

Research paper thumbnail of The molecular chaperones, calnexin and calreticulin, studies on function and mechanism of action

Research paper thumbnail of Studies on Insulin Receptor in Xenopus Laevis Oocytes

Mina Falcone fer her friendship, support and interest. I could always count on her technical advi... more Mina Falcone fer her friendship, support and interest. I could always count on her technical advice, but more importantly I could count on her honest opinion. Jason Young, Gil Abisdris, Josie Ursini and all the people who passed through the Andrews lab for many useful discussions, both scientific and otherwise, and especially for their friendship. Paul MacPherson for his patience, love and support. Without him I would be computer illiterate and often stranded at 3 a.m. without transportation home. My sister Helen, my niece Erin, my mother Panagiota and Jim and Mary MacPherson for their love and support over the years.

Research paper thumbnail of The 3 ' untranslated region of bovine preprolactin contains a transferable non-poly(A) mRNA sequence that prolongs

Research paper thumbnail of GTI-2040, an antisense agent targeting the small subunit component (R2) of human ribonucleotide reductase, shows potent antitumor activity against a variety of tumors

Cancer research, 2003

GTI-2040 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the ... more GTI-2040 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the R2 small subunit component of human ribonucleotide reductase. In vitro studies using a number of human tumor cell lines have demonstrated that GTI-2040 decreases mRNA and protein levels of R2 in a sequence- and target-specific manner. In vivo studies have shown that GTI-2040 significantly inhibits growth of human colon tumors (adenocarcinoma), pancreatic tumors (adenocarcinoma), liver tumors, lung tumors, breast tumors (adenocarcinoma), renal tumors, ovarian tumors (adenocarcinoma), melanoma, brain glioblastoma-astrocytoma, prostatic tumors, and cervical tumors in nude and/or severe combined immunodeficient mice. Antitumor effects were not observed with an oligonucleotide containing four mismatches to the R2 sequence or with a scrambled sequence containing the same base content but not complementary to R2. This suggests that an antisense mechanism is responsible for the in vivo observati...

Research paper thumbnail of Anti-proliferative and anti-tumor effects of antisense oligonucleotide GTI-2601 targeted against human thioredoxin

Anti Cancer Drugs, Mar 1, 2006

Human thioredoxin has been implicated in cancer as a growth stimulator through regulation of DNA ... more Human thioredoxin has been implicated in cancer as a growth stimulator through regulation of DNA replication and growth factor activity, as a modulator of transcription factor activity, and as an inhibitor of apoptosis. In the present study, the steady-state level of thioredoxin protein was examined in a number of cancer cell lines. Interestingly, thioredoxin expression is elevated in a variety of human tumor cell lines compared with normal cell lines. The altered expression of thioredoxin in tumor cells suggests it may be a target in the development of novel therapeutic agents for the treatment and prevention of cancer. Further to this possibility, 26 phosphorothioate antisense oligodeoxynucleotides (PS-AS-ODNs) were evaluated for the ability to inhibit thioredoxin expression in cell culture. One PS-AS-ODN, GTI-2601, specifically reduced the levels of thioredoxin mRNA and protein, exhibited potent anti-proliferative effects on colony formation in vitro, and had anti-tumor effects in human tumor xenograft mouse models in vivo. Sequence-specific decreases in thioredoxin expression levels were accompanied by significant suppression of tumor growth in mice. Taken together, these data suggest that thioredoxin may be a useful target for developing PS-AS-ODNs as drug candidates against human cancer.

Research paper thumbnail of Adenovirus-Mediated Ribonucleotide Reductase R1 Gene Therapy of Human Colon Adenocarcinoma

Clinical Cancer Research, Oct 1, 2003

Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their ... more Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides for DNA synthesis. Ribonucleotide reductase is a multisubunit complex containing two polypeptides, R1 and R2. In addition to catalytic and allosteric regulatory functions, the R1 subunit appears to act as a novel tumor suppressor. Previous studies demonstrated that overexpression of mouse R1 resulted in suppression of tumorigenicity and metastatic potential, whereas expression of antisense RNA, complementary to R1 mRNA, increased anchorage-independent growth of ras-transformed NIH 3T3 cells. The current study investigated the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1).

Research paper thumbnail of Oligosaccharide Binding Characteristics of the Molecular Chaperones Calnexin and Calreticulin †

Biochemistry Usa, Mar 10, 1998

Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their... more Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. We compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, we show that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibited the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. We also examined the effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin. Whereas oligosaccharide binding was only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogated oligosaccharide binding, an effect that was largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins.

Research paper thumbnail of Optimization of the PAXgene TM Blood RNA Extraction System for Gene Expression Analysis of Clinical Samples

J Clin Lab Anal, 2005

One major problem associated with collecting whole blood from patients for use as a source of RNA... more One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene TM blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene TM to stabilize RNA in whole blood; however, RNA extracted by the PAXgene TM method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene TM protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene TM is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Research paper thumbnail of Peptide presentation by MHC class I molecules

Trends in cell biology, 1996

The presentation of peptides by class I histocompatibility molecules plays a central role in the ... more The presentation of peptides by class I histocompatibility molecules plays a central role in the cellular immune response to virally infected or transformed cells. The main steps in this process include the degradation of both self and 'foreign' proteins to short peptides in the cytosol, translocation of peptides into the lumen of the endoplasmic reticulum, binding of a subset of peptides to assembling class I molecules and expression of class-I-peptide complexes at the cell surface for examination by cytotoxic T cells. A molecular understanding of most of these steps is emerging, revealing a remarkable coordination between the processes of peptide translocation, delivery and binding to class I molecules.

Research paper thumbnail of The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules

The EMBO journal, 1996

Calnexin, a membrane protein of the endoplasmic reticulum, is generally thought to function as a ... more Calnexin, a membrane protein of the endoplasmic reticulum, is generally thought to function as a molecular chaperone, based on indirect or correlative evidence. To examine calnexin's functions more directly, we reconstituted the assembly of class I histocompatibility molecules in the absence or presence of calnexin in Drosophila melanogaster cells. Calnexin enhanced the assembly of class I heavy chains with beta 2-microglobulin as much as 5-fold. The improved assembly appeared largely due to more efficient folding of heavy chains, as evidenced by increased reactivity with a conformation-sensitive monoclonal antibody and by a reduction in the level of aggregates. Similar findings were obtained in mouse or human cells when the interaction of calnexin with class I heavy chains was prevented by treatment with the oligosaccharide processing inhibitor castanospermine. The ability of calnexin to facilitate castanospermine. The ability of calnexin to facilitate heavy chain folding and t...

Research paper thumbnail of GTI-2501, an antisense agent targeting R1, the large subunit of human ribonucleotide reductase, shows potent anti-tumor activity against a variety of tumors

International journal of oncology, 2006

GTI-2501 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of R1, ... more GTI-2501 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of R1, the large subunit of ribonucleotide reductase (RNR). In vitro studies, have demonstrated that GTI-2501 decreases mRNA and protein levels of R1 in a sequence-specific and dose-dependent manner. Furthermore, GTI-2501 inhibits the growth of human lung, liver, ovary, brain, melanoma, breast and pancreatic tumor cells in colony forming assays. In vivo studies have shown that GTI-2501 significantly inhibits growth of human colon, pancreas, lung, breast, renal, ovarian, melanoma, brain glioblastoma-astrocytoma, and prostatic tumors in CD-1 nude, Balb/c nude and/or SCID mice. GTI-2501 treatment caused total regression of human breast and renal tumor xenografts in mice. These effects are not observed with a scrambled control oligonucleotide containing the same base content but not complementary to R1. GTI-2501 specifically inhibits metastasis of human melanoma cells to the lungs in CD-1 athymic n...

Research paper thumbnail of GTI-2040, an antisense agent targeting the small subunit component (R2) of human ribonucleotide reductase, shows potent antitumor activity against a variety of tumors

Cancer research, 2003

GTI-2040 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the ... more GTI-2040 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the R2 small subunit component of human ribonucleotide reductase. In vitro studies using a number of human tumor cell lines have demonstrated that GTI-2040 decreases mRNA and protein levels of R2 in a sequence- and target-specific manner. In vivo studies have shown that GTI-2040 significantly inhibits growth of human colon tumors (adenocarcinoma), pancreatic tumors (adenocarcinoma), liver tumors, lung tumors, breast tumors (adenocarcinoma), renal tumors, ovarian tumors (adenocarcinoma), melanoma, brain glioblastoma-astrocytoma, prostatic tumors, and cervical tumors in nude and/or severe combined immunodeficient mice. Antitumor effects were not observed with an oligonucleotide containing four mismatches to the R2 sequence or with a scrambled sequence containing the same base content but not complementary to R2. This suggests that an antisense mechanism is responsible for the in vivo observati...

Research paper thumbnail of Adenovirus-mediated ribonucleotide reductase R1 gene therapy of human colon adenocarcinoma

Clinical cancer research : an official journal of the American Association for Cancer Research, 2003

Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their ... more Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides for DNA synthesis. Ribonucleotide reductase is a multisubunit complex containing two polypeptides, R1 and R2. In addition to catalytic and allosteric regulatory functions, the R1 subunit appears to act as a novel tumor suppressor. Previous studies demonstrated that overexpression of mouse R1 resulted in suppression of tumorigenicity and metastatic potential, whereas expression of antisense RNA, complementary to R1 mRNA, increased anchorage-independent growth of ras-transformed NIH 3T3 cells. The current study investigated the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1). Recombinant viruses were constructed by FLP-mediated site-specific recombination and demonstrated high infectivity of a human colon carcinoma cell line (Colo320 HRS), as assessed by expression of a viral encoded beta-G...

Research paper thumbnail of GTI-2040 displays cooperative anti-tumor activity when combined with interferon α against human renal carcinoma xenografts

International Journal of Oncology, 1992

, an antisense oligonucleotide targeting the small subunit of ribonucleotide reductase, acts as a... more , an antisense oligonucleotide targeting the small subunit of ribonucleotide reductase, acts as an anti-tumor agent in animal models of human cancer. In the present study, the anti-tumor activity of GTI-2040, in combination with interferon · (IFN·) was investigated against human renal cell carcinoma tumors xenografted into mice. The human renal cell carcinoma cell lines, Caki-1 and A498 were sensitive to IFN· both in vitro and when implanted into mice. In combination with GTI-2040 there were cooperative effects at intermediate doses of the two agents and complete tumor regression at higher combination doses. A control oligonucleotide was not effective as a monotherapy and did not improve the efficacy of IFN·. The effect of combination treatment on apoptosis and proliferation of tumor cells, isolated from xenografted tumors, was examined by histochemistry. GTI-2040 increased the percentage of cells undergoing apoptosis with a concomitant decrease in proliferation. IFN· alone had no effect but in combination with GTI-2040 resulted in increased apoptosis and decreased proliferation compared to GTI-2040 alone. Taken together these results expand the potential clinical applications of GTI-2040 to include combination therapy with IFN·.

Research paper thumbnail of Analysis of ribonucleotide reductase M2 mRNA levels in patient samples after GTI-2040 antisense drug treatment

Oncology Reports, 2006

This study describes the development of a rapid and practical real-time RT-PCR method to quantify... more This study describes the development of a rapid and practical real-time RT-PCR method to quantify ribonucleotide reductase M2 (RRM2) mRNA in tumor and peripheral white blood cells (WBCs) from patients treated with GTI-2040, an antisense drug currently in clinical trials. In order to assess target down-regulation by GTI-2040, RRM2 mRNA expression levels were analyzed in pre-and post-treatment samples from a phase II clinical trial of GTI-2040 combined with capecitabine in patients with metastatic breast cancer. Target gene RRM2 mRNA levels were evaluated using quantitative RT-PCR method: real-time PCR (TaqMan ® ) with fluorescein labeled probes on an ABI 7900HT instrument, with additional post-processing of the data to adjust for differences in total RNA in-put across the samples. Data are presented from a patient for whom both biopsy and PBMC samples were available, demonstrating applicability of this reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of mRNAs for RRM2 in human WBC and tissue samples. By providing quantitative measurement of changes in target gene expression, this method may provide an opportunity to determine the correlation between target response to GTI-2040 antisense and clinical response in patients. Furthermore this assay may assess whether WBC samples are an appropriate surrogate tissue for approximating target down-regulation in the tumor.

Research paper thumbnail of NC381, a Novel Anticancer Agent, Arrests the Cell Cycle in G0-G1 and Inhibits Lung Tumor Cell Growth in Vitro and in Vivo

Journal of Pharmacology and Experimental Therapeutics, 2003

Although clotrimazole (CLT), an antifungal drug, inhibits tumor cell proliferation and angiogenes... more Although clotrimazole (CLT), an antifungal drug, inhibits tumor cell proliferation and angiogenesis, its clinical application is hampered by significant hepatotoxicity due to the presence of an imidazole moiety. In our attempts to develop CLT analogs that are devoid of imidazole and are as efficacious as CLT, one pharmacophore designated NC381 was generated and shown to inhibit tumor cell growth via a mechanism similar to that of CLT. In vitro, treatment of NCI-H460 nonsmall cell lung cancer (NSCLC) cells with NC381 inhibited growth in a time-dependent manner. Flow cytometric analysis demonstrated that the decrease in cell growth was associated with inhibition of cell cycle progression at the G(1)-S phase transition, resulting in G(0)-G(1) arrest. There was a concomitant inhibition of cyclin D1 expression and subsequent reduction in the formation of the cyclin D1-CDK4 complex. Consistent with a decrease in the cyclin D1-CDK4 complex, NC381 treatment resulted in significant inhibition of pRb phosphorylation. There also were changes in the activity of cell cycle-related proteins, including p16(Ink4) and p27(Kip1). Together, these results are consistent with a model in which NC381 arrests cell cycle progression via inhibition of the pathway that promotes exit from the G(1) phase of the cell cycle. Furthermore, the clinical applicability of NC381 was evaluated in an in vivo murine xenograft model of human NSCLC (NCI-H460). NC381 treatment resulted in significant inhibition of tumor growth. Given the poor prognosis and the limited treatment options available, the present results underscore the potential of NC381 in the treatment of human NSCLC.

Research paper thumbnail of Optimization of the PAXgene™ blood RNA extraction system for gene expression analysis of clinical samples

Journal of Clinical Laboratory Analysis, 2005

One major problem associated with collecting whole blood from patients for use as a source of RNA... more One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene TM blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene TM to stabilize RNA in whole blood; however, RNA extracted by the PAXgene TM method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene TM protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene TM is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Research paper thumbnail of The Molecular Chaperone Calnexin Binds Glc(1)Man(9)GlcNAc(2) Oligosaccharide as an Initial Step in Recognizing Unfolded Glycoproteins

Journal of Biological Chemistry, 1995