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Biomedical subjects

S Pepe

Publications and source records attributed to S Pepe.

At least 37 records · Page 2Linked to original sources
PubMed1999

Antisense DNA-targeting protein kinase A-RIA subunit: a novel approach to cancer treatment.

Enhanced expression of the RIa subunit of cAMP-dependent protein kinase type I (PKA-I) has been shown during carcinogenesis, in human cancer cell lines and in primary tumors. We demonstrate that the sequence-specific inhibition of RIa gene expression by antisense oligonucleotides results in the differentiation of leukemia cells and growth arrest of cancer cells of epithelial origin and tumors in mice. The loss of RI by the antisense results in rapid increase in the half-life of the competitor molecule, RII protein, via its stabilization in a holoenzyme complex (PKA-II) that insures depletion of PKA-I and sustained inhibition of tumor growth. RI antisense, which restrains tumor cell growth by turning on the signals for blockade of tumor cell survival, namely blockade of the tyrosine kinase signaling, cell cycle deregulation and apoptosis, provides a single gene-targeting approach to treatment of cancer.

Animals↗
PubMed1999

Key role of the cyclin-dependent kinase inhibitor p27kip1 for embryonal carcinoma cell survival and differentiation.

Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hybrid polar compounds, which induce differentiation in a variety of transformed cells including human embryonal carcinoma cells. Therefore, HMBA has been used in the differentiation therapy of cancer for patients with both hematological and solid malignancies. Upon HMBA treatment, the embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes terminal differentiation. Here we demonstrate that growth arrest and differentiation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyclin E/CDK2 complexes and suppression of kinase activity associated to cyclin E/CDK2 (but not to cyclin D3/CDK4). When HMBA differentiation was induced in the presence of p27 antisense oligonucleotides, NT2/D1 cells failed to arrest growth properly and, in parallel with the reduction of the anti-apoptotic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a marked reduction in the growth potential of these cells and partially reproduced HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-53 expression). Expression of p21 induced growth arrest but not differentiation. Likewise, inhibition of CDK2 by transfection of a dominant negative CDK2 in NT2/D1 cells or treatment with the kinase inhibitor olomucine induced growth arrest but not differentiation. Therefore, we propose that p27 represents a crucial molecule in HMBA signaling that cannot be replaced by p21. Furthermore, the results obtained with CDK2 inhibitors demonstrate that the block of CDK2 activity is sufficient for growth arrest but not for cell differentiation and suggest that, at least in these cells, growth arrest and differentiation are regulated by two overlapping but different pathways.

Acetamides↗
PubMed1999

Mitochondrial DNA deletions in human cardiac tissue show a gross mosaic distribution.

The variability of mitochondrial DNA (mtDNA) deletional patterns has been investigated in adjacent slices of human heart atrium. Using quantitative PCR we found differential abundances of one particular mtDNA deletion, that of 4977 bp (mtDNA4977), among sets of adjacent slices of right atrial trabeculae pectinatae from 10 subjects. Some subjects had relatively constant abundance of mtDNA4977 among the tissue slices, while others covered a wide range. A qualitative PCR procedure was used to visualize the patterns of multiple deletions within an 8.64-kb segment of the mtDNA genome, in the same set of atrial trabeculae samples. Some subjects showed completely different multiple deletional patterns in each of the trabeculae slices analyzed. There was no correlation between the variation of the abundance of mtDNA4977 and that of the multiple deletions. The results are consistent with the notion that the occurrence of mtDNA deletions during aging is a random process, involving their production throughout the lifetime of an individual. In this view, the patterns of new deletions are superimposed on those already accumulated by propagation and segregation of mutations formed earlier in life.

Adult↗
PubMed1999

Coenzyme Q10 improves the tolerance of the senescent myocardium to aerobic and ischemic stress: studies in rats and in human atrial tissue.

The inferior recovery of cardiac function after interventional cardiac procedures in elderly patients compared to younger patients suggests that the aged myocardium is more sensitive to stress. We report two studies that demonstrate an age-related deficit in myocardial performance after aerobic and ischemic stress and the capacity of CoQ10 treatment to correct age-specific diminished recovery of function. In Study 1 the functional recovery of young (4 mo) and senescent (35 mo) isolated working rat hearts after aerobic stress produced by rapid electrical pacing was examined. After pacing, the senescent hearts, compared to young, showed reduced recovery of pre-stress work performance. CoQ10 pretreatment (daily intraperitoneal injections of 4 mg/kg CoQ10 for 6 weeks) in senescent hearts improved their recovery to match that of young hearts. Study 2 tested whether the capacity of human atrial trabeculae (obtained during surgery) to recover contractile function, following ischemic stress in vitro (60 min), is decreased with age and whether this decrease can be reversed by CoQ10. Trabeculae from older individuals (> or = 70 yr) showed reduced recovery of developed force after simulated ischemia compared to younger counterparts (< 70 yr). Notably, this age-associated effect was prevented in trabeculae pretreated in vitro (30 min at 24 degrees C) with CoQ10 (400 MicroM). We measured significantly lower CoQ10 content in trabeculae from > or = 70 yr patients. In vitro pretreatment raised trabecular CoQ10 content to similar levels in all groups. We conclude that, compared to younger counterparts, the senescent myocardium of rats and humans has a reduced capacity to tolerate ischemic or aerobic stress and recover pre-stress contractile performance, however, this reduction is attenuated by CoQ10 pretreatment.

Aerobiosis↗
PubMed1999

Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor.

We have demonstrated that interferon-alpha2-recombinant (IFNalpha) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNalpha underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNalpha enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNalpha. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNalpha. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNalpha and targeted on the stress pathway terminal kinases.

Apoptosis↗
PubMed1999

Mitochondria in heart ischaemia and aging.

We have examined the effect of dietary polyunsaturated fatty acids (PUFAs) upon mitochondrial Ca2+ content and dehydrogenase activation in the rat heart. Diets were either a conventional low-fat chow (Ref) or were rich in n-3 PUFAs from fish oils (n-3) or n-6 PUFAs from animal fat (n-6). We found that the n-3 diet minimized the rise in mitochondrial Ca2+ seen in response to positive inotropic intervention with noradrenaline, and also minimized the activation of pyruvate dehydrogenase, which is Ca2+ dependent. As the work output of all three groups of hearts was the same, this observation may explain the previous finding of increased thermodynamic efficiency of the n-3 heart relative to the n-6 heart. When hearts were subjected to low-flow ischaemia (15 min), followed by 5 min of reperfusion, increases of mitochondrial Ca2+ were less in the n-3 group than in the n-6 group. In more prolonged ischaemia and reperfusion, n-3 feeding may confer protection against mitochondrial Ca2+ overload, opening of the permeability transition pore and cell death. Notably, the effects of n-3 feeding on mitochondrial functioning were most apparent in hearts from senescent rats (23 months). This is consistent with our finding that the decrease in mitochondrial membrane cardiolipin content, and increase in phosphatidylcholine, which occurred with aging in the Ref and n-6 groups, was totally prevented by n-3 feeding. Thus there are a number of reasons to regard an n-3-rich diet as being protective of the heart in aging mammals.

Aging↗
PubMed1999

PUFA and aging modulate cardiac mitochondrial membrane lipid composition and Ca2+ activation of PDH.

Aberrations in cell Ca2+ homeostasis have been known to parallel both changes in membrane lipid composition and aging. Previous work has shown that the lowered efficiency of work performance, which occurs in isolated hearts from rats fed a diet rich in n-6 polyunsaturated fatty acids (PUFA), relative to those fed n-3 PUFA, could be raised by mitochondrial (Mito) Ca2+ transport inhibition. We tested whether, after Ca2+-dependent stress, the Ca2+-dependent activation of pyruvate dehydrogenase (PDHA/PDHTotal) and Mito Ca2+ cycling could be manipulated by varying the ratio of n-3 to n-6 PUFA in Mito membranes in young (6 mo) and aged (24 mo) isolated rat hearts treated to n-3 or n-6 PUFA-rich diet. Inotropic stimulation by 1 microM norepinephrine (NE) of 24-mo n-6 PUFA-rich hearts elevated total Mito Ca2+ content 38% more than in 6-mo hearts (P < 0. 05). However, both the NE-induced rise in Mito Ca2+ and the difference in response between 6- and 24-mo hearts were partially abolished by n-3 PUFA treatment. NE increased the fractional activation of PDH by 44% above control levels in the 6-mo group compared with 49% in the 24-mo group after n-6 PUFA diet. However, NE stimulation of PDHA was attenuated by n-3 PUFA diet, attaining values only 29 and 23% above control levels in 6- and 24-mo mitochondria, respectively (P < 0.05). Global ischemia and reperfusion (I/R) in n-6 PUFA hearts gave rise to higher levels of total Mito Ca2+ concentration (P < 0.0001) and PDHA (P < 0.0001) compared with n-3 PUFA. Ruthenium red (3.4 microM) abolished the effects of I/R in all groups. With aging, heart Mito membrane phosphatidylcholine was increased after n-6 PUFA-rich diet (by approximately 15%, P < 0.05), whereas cardiolipin and n-3 PUFA content were diminished by 31% (P < 0.05) and 73% (P < 0.05), respectively. These effects were prevented by n-3 PUFA-rich diet. The present study, by directly manipulating the cardiac Mito membrane n-3-to-n-6 PUFA ratio, shows that the activation of Ca2+-dependent PDH can be augmented when the n-3-to-n-6 PUFA ratio is low (n-6 PUFA-rich diet; 24-mo hearts) or attenuated when this ratio is relatively high (n-3 PUFA-rich diet). We propose that one of the consequences of dietary-induced manipulation of membrane phospholipids and PUFAs may be the altered flux of Ca2+ across the Mito membrane and thus altered intramitochondrial Ca2+-dependent processes.

Aging↗
PubMed1999

Cooperative inhibitory effect of novel mixed backbone oligonucleotide targeting protein kinase A in combination with docetaxel and anti-epidermal growth factor-receptor antibody on human breast cancer cell growth.

Type I protein kinase A (PKAI) is overexpressed in the majority of human tumors and plays a relevant role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of PKAI by antisense oligonucleotides targeting its RIalpha regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. We have recently shown that a mixed backbone oligonucleotide targeting RIalpha can cooperatively inhibit human cancer cell growth when combined with selected cytotoxic drugs. In the present study, we have used HYB 165, a novel DNA/RNA hybrid mixed backbone oligonucleotide that exhibits improved pharmacokinetic and bioavailability properties in vivo and is presently undergoing Phase I trials. We have shown that HYB 165 exhibits a dose-dependent inhibitory effect on ZR-75-1 cells and a cooperative activity with docetaxel, a cytotoxic drug active in breast cancer. The antiproliferative activity is accompanied by increased apoptosis, as compared with each single agent. On the basis of our previous demonstration of a structural and functional relation between PKAI and epidermal growth factor receptor, we have performed a double blockade of these pathways using HYB 165 in combination with monoclonal antibody (MAb) C225, an anti-epidermal growth factor receptor chimeric MAb. The two compounds determined a cooperative growth inhibitory effect on ZR-75-1 cells and increased apoptosis. To study whether different biological agents and cytotoxic drugs can interact together, low doses of HYB 165, MAb C225, and docetaxel were combined causing an even greater cooperative effect toward growth inhibition. Finally, we have demonstrated that each single agent is able to induce bcl-2 phosphorylation and that the three agents, used in combination at suboptimal doses, determine a greater degree of bcl-2 phosphorylation and cause apoptosis of the majority of ZR-75-1 cells. These findings provide the basis for a novel strategy of treatment of breast cancer patients because both HYB 165 and MAb C225 are presently under clinical evaluation.

Antibodies, Monoclonal↗
PubMed1999

Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225.

Epidermal growth factor (EGF)-related proteins such as transforming growth factor alpha (TGF-alpha) control cancer cell growth through autocrine and paracrine pathways. Overexpression of TGF-alpha and/or its receptor (EGFR) has been associated with a more aggressive disease and a poor prognosis. The blockade of EGFR activation has been proposed as a target for anticancer therapy. Monoclonal antibody (MAb) C225 is an anti-EGFR humanized chimeric mouse MAb that is presently in Phase II clinical trials in cancer patients. Previous studies have suggested the potentiation of the antitumor activity of certain cytotoxic drugs, such as cisplatin and doxorubicin, in human cancer cell lines by treatment with anti-EGFR antibodies. We have evaluated in human ovarian, breast, and colon cancer cell lines, which express functional EGFR, the antiproliferative activity of MAb C225 in combination with topotecan, a cytotoxic drug that specifically inhibits topoisomerase I and that has shown antitumor activity in these malignancies. A dose-dependent supraadditive increase of growth inhibition in vitro was observed when cancer cells were treated with topotecan and MAb C225 in a sequential schedule. In this respect, the cooperativity quotient, defined as the ratio between the actual growth inhibition obtained by treatment with topotecan followed by MAb C225 and the sum of the growth inhibition achieved by each agent, ranged from 1.2 to 3, depending on drug concentration and cancer cell line. Treatment with MAb C225 also markedly enhanced apoptotic cell death induced by topotecan. For example, in GEO colon cancer cells, 5 nM topotecan, followed by 0.5 microg/ml MAb C225, induced apoptosis in 45% cells as compared with untreated cells (6%) or to 5 nM topotecan-treated cells (22%). Treatment of mice bearing established human GEO colon cancer xenografts with topotecan or with MAb C225 determined a transient inhibition of tumor growth because GEO tumors resumed the growth rate of untreated tumors at the end of the treatment period. In contrast, an almost complete tumor regression was observed in all mice treated with the two agents in combination. This determined a prolonged life span of the mice that was significantly different as compared with controls (P < 0.001), to MAb C225-treated group (P < 0.001), or to the topotecan-treated group (P < 0.001). All mice of the topotecan plus MAb C225 group were the only animals alive 14 weeks after tumor cell injection. Furthermore, 20% of mice in this group were still alive after 19 weeks. The combined treatment with MAb C225 and topotecan was well tolerated by mice with no signs of acute or delayed toxicity. These results provide a rationale for the evaluation of the anticancer activity of the combination of topoisomerase I inhibitors and anti-EGFR blocking MAbs in clinical trials.

Animals↗
PubMed1999

Measurement of neovascularization is an independent prognosticator of survival in node-negative breast cancer patients with long-term follow-up.

We measured neovascularization, epidermal growth factor receptor, and c-erbB-2 expression in a consecutive series of 233 surgically resected axillary lymph node-negative breast cancer patients with a long-term follow-up to define the usefulness of these parameters as independent prognostic indicators of overall survival (OAS). Microvessel count (MVC), as a measure of neovascularization, was determined using a monoclonal antibody against human factor VIII-related antigen. The median MVC of 20 (range, 4-76) was used as a cutoff value for discriminating between low and high vascularized tumors. Epidermal growth factor receptor and c-erbB-2 expression were evaluated by immunohistochemistry. Tumors were considered positive if >10% of the cells showed specific membrane staining. OAS curves were estimated by the Kaplan-Meier method. The independent prognostic effect of each variable was determined with the Cox proportional hazards model. High MVC (P = 0.04), high nuclear grade (P = 0.005), and high S-phase (P = 0.02) significantly affected OAS at univariate analysis. In a Cox multivariate analysis, the characteristics with an independent prognostic effect on OAS were: MVC (relative hazard, 2.12; 95% confidence interval, 1.18-3.81; P = 0.01) and nuclear grade (relative hazard, 2.83; 95% confidence interval, 1.12-7.17; P = 0.01). These results demonstrate that quantification of neovascularization adds useful independent prognostic information on survival in node-negative breast cancer patients with long-term follow-up.

Adult↗
PubMed1998

Cooperative inhibition of renal cancer growth by anti-epidermal growth factor receptor antibody and protein kinase A antisense oligonucleotide.

BACKGROUND: The expression of epidermal growth factor receptor (EGFR) and type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is associated with neoplastic transformation. By use of human renal cancer cell lines (i.e., 769-P, ACHN, A498, and SW839), we investigated the antiproliferative activity and the antitumor activity of an anti-EGFR humanized chimeric mouse monoclonal antibody, MAb C225, and a novel mixed backbone 18-mer antisense oligonucleotide, HYB 190, that targets expression of the RIalpha regulatory subunit of PKAI. METHODS: The antiproliferative activity of MAb C225 and oligonucleotide HYB 190, alone or in combination, on different renal cancer cell lines was determined by monitoring cell growth in soft agar. In addition, the induction of apoptosis by treatment with the anti-EGFR antibody and/or antisense PKAI oligonucleotides was evaluated by flow cytometric analysis of fragmented DNA. The antitumor activity of MAb C225 and oligonucleotide HYB 190 was determined in athymic mice bearing established ACHN tumor xenografts. Cell proliferation and tumor growth data were evaluated for statistical significance using Student's t test; reported P values are two-sided. RESULTS: MAb C225 and oligonucleotide HYB 190 inhibited colony formation in soft agar in a dose-dependent manner for all renal cancer cell lines tested. We observed a potentiation of growth inhibition and induction of apoptosis when 769-P cells and ACHN cells were treated with both agents. Combination treatment with MAb C225 and oligonucleotide HYB 190 caused regression of ACHN tumor xenografts, whereas single-agent treatment only delayed tumor growth. CONCLUSION: The combination of anti-EGFR MAb C225 and ited cooperative antiproliferative effects and cooperative antitumor effects on EGFR and PKAI-expressing human renal cancer cell lines.

Animals↗
PubMed1998

Mechanism of cardioprotective effect of orotic acid.

The pyrimidine base, orotic acid (OA), markedly improves the function of recently infarcted hearts subjected to global ischemia. The mechanism of cardiac action of OA is unclear, but it has been proposed that OA acts by correcting a relative deficiency of nucleotide precursors required for RNA synthesis in the stressed myocardium or by improving myocardial energy supply. The aim of this study was to investigate the mechanism of action of OA by (1) determining whether a high dose of OA can raise the concentration of pyrimidine metabolites in plasma, liver, and heart; (2) examining the effects of OA on adenine nucleotide (AN) concentrations in normal and infarcted hearts, before and after global ischemia; and (3) determining the effect of uridine, an important metabolite of OA, on myocardial energy metabolism. Three studies were performed: (1) The time course of changes in tissue and plasma concentrations of pyrimidine compounds was examined in unoperated rats after the administration of 100 mg/kg OA. (2) Rats were given OA (30 mg/kg/d) for 2 days after experimental infarction, and tissue and plasma pyrimidine concentrations were examined; the hearts were removed for perfusion in the isolated working rat heart model (37 degrees C), subjected to 30 minutes of global ischemia, and recovery of function was assessed. AN content was assessed in the noninfarcted myocardium before and after ischemia. Isolated hearts were subjected to 30 minutes of hypoxic perfusion and the effect of adding 17 microM uridine to the perfusate was examined. Study 1 showed that OA administration produced an increase in hepatic uridine and cytidine, followed by increased plasma uridine and cytidine (cytidine, +55%, P < 0.001; uridine, +124%, P = 0.011). Myocardial uracil nucleotides increased temporarily after 4 hours (+21%, P < 0.01). In infarcted hearts after 2 days of OA administration, there were no significant changes in myocardial uracil or cytosine nucleotides or total RNA. Infarction significantly reduced functional recovery after global ischemia (sham = 62%; infarct = 26% of preischemic level; P < 0.05). OA improved the recovery of preischemic function by 133% (P < 0.05) in infarcted, but not sham-operated, hearts. Preischemic ATP and total adenine nucleotides (TAN) were decreased in the surviving myocardium of infarcted hearts (ATP reduced from 21.7 +/- 0.8 to 14.7 +/- 0.7 mumol/g dry wt, P < 0.001; TAN decreased from 30.3 +/- 0.8 to 22.4 +/- 1.1 mumol/g dry wt, P < 0.001). OA treatment prevented these reductions. Study 3 showed that uridine improved myocardial ATP and TAN levels, and decreased purine loss in hypoxic hearts. The increased AN levels were accompanied by evidence of enhancement of anerobic glycolysis. We conclude: (1) That OA acts on the heart via the liver by increasing the availability of plasma uridine and cytidine. (2) Uridine is capable of increasing myocardial ATP production by stimulating anerobic glycolysis. (3) OA treatment improves tolerance to global ischemia in infarcted but not normal hearts by preventing depletion of AN in the surviving myocardium.

Adenine Nucleotides↗
PubMed1998

Desferioxamine increases iron depletion and apoptosis induced by ara-C of human myeloid leukaemic cells.

We investigated whether changes in iron metabolism and the transferrin receptor (TRF-R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara-C). Treatment with 100 nM ara-C for 48h reduced thymidine uptake and increased the surface expression of the TRF-R on leukaemic blasts derived from 13/16 (81%) patients and on the HL-60 and U-937 cell lines. Whereas intracellular non-haem iron was strongly depleted 24 h after ara-C addition, TRF-R up-regulation and recovery of intracellular non-haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara-C and the iron chelator desferioxamine (DSF) on the growth of HL-60 and U-937 cells. We found that desferioxamine strongly potentiated the effects of ara-C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara-C and an iron chelator in terms of antileukaemic effects.

Acute Disease↗
PubMed1997

Synergistic inhibition of growth and induction of apoptosis by 8-chloro-cAMP and paclitaxel or cisplatin in human cancer cells.

8-Chloro-cAMP (8-Cl-cAMP) is a novel agent that is able to inhibit the growth of a wide variety of cancer cell types in vitro and in vivo and, at doses devoid of toxicity, to achieve plasma concentrations in cancer patients in a range effective for cancer cell growth inhibition. In this study, we have demonstrated that 8-Cl-cAMP, at a dose causing mild or no growth inhibition, synergistically increased the growth-inhibitory effect of paclitaxel or cisplatin in a wide series of cell lines including human breast, lung, ovary, colon, and head carcinomas and melanoma. A similar effect was also observed with another taxane, docetaxel, and with the platinum-derivative carboplatin. 8-Cl-cAMP also markedly enhanced apoptotic cell death induced by each cytotoxic drug. A cooperative antitumor effect was also observed in vivo, because treatment with paclitaxel followed by 8-Cl-cAMP markedly inhibited the growth of GEO human colon cancer xenografts as compared to paclitaxel alone without signs of toxicity. These data demonstrate that 8-Cl-cAMP synergistically increases the antiproliferative activity of taxanes and platinum-derived compounds and provide a rationale to use 8-Cl-cAMP in combination with taxanes and platinum-derived compounds.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A.

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIalpha regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

Animals↗
PubMed1997

Anti-sense oligonucleotides directed against EGF-related growth factors enhance anti-proliferative effect of conventional anti-tumor drugs in human colon-cancer cells.

We have demonstrated that anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against the EGF-like growth factors CRIPTO (CR), amphiregulin (AR) or transforming-growth-factor-alpha(TGFalpha) mRNA, are equipotent in their ability to inhibit the growth of human colon-carcinoma GEO cells. In this study, we evaluated the effect of combinations of these AS S-oligos and conventional anti tumor drugs, such as 5-fluorouracil (5-FU), adriamycin (ADR), mitomycin C (MIT) and cis-platinum (CDDP), on GEO cell growth. Dose-dependent growth inhibition was observed by treatment either with AS S-oligos or with anti-tumor drugs, using a clonogenic assay. Furthermore, an additive growth inhibitory effect occurred when GEO cells were exposed to the AS S-oligos after treatment with different concentrations of either 5-FU, MIT, ADR or CDDP. For example, treatment of GEO cells with a combination of low concentrations of 5-FU and any of the 3 AS S-oligos resulted in up to 70% growth inhibition. However, treatment of GEO cells with AS S-oligos before exposure to 5-FU or CDDP resulted in reduced efficacy of both drugs. Flow-cytometric analysis of DNA content demonstrated that treatment with the AS S-oligos caused a slight reduction of the percentage of cells in the S-phase of the cell cycle. These data suggest that combinations of AS S-oligos directed against EGF-related growth factors and of conventional anti-tumor drugs may result in efficient inhibition of colon-carcinoma cell growth.

Amphiregulin↗
PubMed1997

Expression of teratocarcinoma-derived growth factor-1 (TDGF-1) in testis germ cell tumors and its effects on growth and differentiation of embryonal carcinoma cell line NTERA2/D1.

The teratocarcinoma-derived growth factor-1 (TDGF-1) gene codes for a 188-aminoacid glycoprotein that shares structural homology with the epidermal growth factor (EGF) family of growth factors. TDGF-1 is highly expressed in the undifferentiated embryonal carcinoma stem cell line NTERA2 clone D1 (NT2/D1) and its expression is downregulated in response to differentiating agents such as retinoic acid (RA) and hexamethylen-bisacetamide (HMBA). To assess the role of TDGF-1 in the onset and/or progression of human germ cell tumors, we analysed TDGF-1 expression by Northern blot and immunostaining in a panel of 59 human germ cell tumors of different histological origins. We show that TDGF-1 expression is markedly elevated in a subset of human testicular germ cell tumors as compared to normal testes. TDGF-1 overexpression occurs in about 100% of tumors with non-seminomatous phenotype, such as embryonal carcinomas and malignant undifferentiated teratocarcinomas. To address the questions of how TDGF-1 (previously called CRIPTO) may affect the growth and/or the differentiation of embryonal carcinoma cells, we have characterized the effects of exogenous recombinant TDGF-1 protein on the proliferation rate and differentiation 'potential of NT2/D1. Exogenous TDGF-1 protein stimulated DNA synthesis and cell proliferation in both undifferentiated and differentiated NT2/D1 cells. However, TDGF-1 protein treatment was unable to block differentiation induced by both RA and HMBA. These results suggest that TDGF-1 growth factor may represent an autocrine growth factor that may be involved in the process of development of testicular neoplasms.

Amino Acid Sequence↗