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F Ciardiello

Publications and source records attributed to F Ciardiello.

At least 55 records · Page 3Linked to original sources
PubMed1998

Down-regulation of type I protein kinase A by transfection of human breast cancer cells with an epidermal growth factor receptor antisense expression vector.

MDA-468 human breast cancer cells overexpress the EGFR and exhibit a functional TGFalpha-EGFR autocrine pathway. Loss of EGFR expression following stable transfection with an antisense EGFR cDNA containing plasmid down-regulates type I cAMP-dependent protein kinase (PKAI) expression with acquisition of cell growth resistance to the PKAI inhibitor 8-Cl-cAMP. These results suggest that PKAI expression and function are controlled by a TGFalpha-EGFR autocrine pathway in human breast cancer cells overexpressing the EGFR.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Epidermal growth factor-related peptides as targets for experimental therapy of human colon carcinoma.

Colorectal carcinomas generally show a poor sensitivity to conventional chemotherapeutics. Therefore, novel therapeutic approaches are required to improve the prognosis of colon cancer patients in advanced stage. Several growth factors are involved in the control of colon carcinoma cell proliferation. In particular, the epidermal growth factor (EGF)-related peptides transforming growth factor-alpha (TGF-alpha), amphi-regulin (AR), and CRIPTO (CR) are frequently overexpressed in human colon carcinomas. It has also been recently demonstrated that they can function as autocrine growth factors in human colon carcinoma cells. In fact, antisense (AS) retroviral expression vectors or AS oligonucleotides directed against TGF-alpha, AR, or CR are able to inhibit growth and transformation of several human colon carcinoma cell lines. These data suggest that the EGF-like growth factors and their receptors offer potential as targets for experimental therapy of human colon carcinoma. This article reviews the most recent findings in this field.

Animals↗
PubMed1998

Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer: amphiregulin and microvessel count are independent prognostic indicators of survival.

We have determined the expression of transforming growth factor alpha (TGF alpha), amphiregulin (AR), CRIPTO, the epidermal growth factor receptor (EGFR), erbB-2, erbB-3, and tumor angiogenesis in a series of 195 patients with stage I-IIIA non-small cell lung cancer (NSCLC) treated with radical surgery to define their usefulness as prognostic indicators of survival. A variable degree of specific staining in cancer cells was observed for the three growth factors and for the three growth factor receptors in the majority of NSCLC patients. A statistically significant association between overexpression of TGF alpha, AR, and CRIPTO was observed. Enhanced expression of AR was significantly correlated with enhanced expression of erbB-2 and advanced T-stage. A direct association was also detected for overexpression of TGF alpha and of erbB-2 or erbB-3, respectively. Sex, tumor size, nodal status, stage, microvessel count, as a measure of neovascularization, and AR overexpression significantly correlated with overall survival at univariate analysis. In a Cox multivariate analysis, the only characteristics with an independent prognostic effect on OAS were microvessel count [relative hazard (RH), 6.61; P < 0.00001), nodal status (RH, 1.59; P = 0.0013), and AR overexpression (RH, 1.72; P = 0.02). These results suggest that evaluation of neoangiogenesis and of certain growth factors, such as AR, can be useful in addition to conventional pathological staging to select high-risk NSCLC patients who may benefit from post-surgical systemic therapies.

Adult↗
PubMed1998

Interactions between the epidermal growth factor receptor and type I protein kinase A: biological significance and therapeutic implications.

Peptide growth factors regulate normal cellular proliferation and differentiation through autocrine and paracrine pathways and are involved in cancer development and progression. Among the endogenous growth factors, the epidermal growth factor (EGF)-related proteins play an important role in the pathogenesis of human cancer. In fact, overexpression of EGF-related growth factors such as transforming growth factor alpha and amphiregulin and/or their specific receptor, the EGF receptor (EGFR), has been detected in several types of human cancers, including breast, lung, and colorectal cancers. Therefore, the blockade of EGFR activation by using anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The cAMP-dependent protein kinase (PKA) is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth and differentiation. Two PKA isoforms with identical catalytic (C) subunits but different cAMP-binding regulatory (R) subunits (defined as RI in PKAI and RII in PKAII) have been identified. Predominant expression of PKAII is found in normal nonproliferating tissues and in growth-arrested cells, whereas enhanced levels of PKAI are detected steadily in tumor cells and transiently in normal cells exposed to mitogenic stimuli. Overexpression of PKAI has been correlated recently with poor prognosis in breast cancer patients. Inhibition of PKAI expression and function by specific pharmacological agents such as the selective cAMP analogue 8-chloro-cAMP (8-Cl-cAMP) induces growth inhibition in various human cancer cell lines in vitro and in vivo. We have provided experimental evidence of a functional cross-talk between ligand-induced EGFR activation and PKAI expression and function. In fact, PKAI is overexpressed and activated following transforming growth factor alpha-induced transformation in several rodent and human cell line models. Furthermore, PKAI is involved in the intracellular mitogenic signaling following ligand-induced EGFR activation. We have shown that an interaction between EGFR and PKAI occurs through direct binding of the RI subunit to the Grb2 adaptor protein. In this respect, PKAI seems to function downstream of the EGFR, and experimental evidence suggests that PKAI is acting upstream of the mitogen-activated protein kinase pathway. We have also demonstrated that the functional interaction between the EGFR and the PKAI pathways could have potential therapeutic implications. In fact, the combined interference with both EGFR and PKAI with specific pharmacological agents, such as anti-EGFR blocking MAbs and cAMP analogues, has a cooperative antiproliferative effect on human cancer cell lines in vitro and in vivo. The antitumor activity of this combination could be explored in a clinical setting because both the 8-Cl-cAMP analogue and the anti-EGFR blocking MAb C225 have entered human clinical trial evaluation. Finally, both MAb C225 and 8-Cl-cAMP are specific inhibitors of intracellular mitogenic signaling that have different mechanisms of action compared with conventional cytotoxic drugs. In this respect, a cooperative growth-inhibitory effect in combination with several chemotherapeutic agents in a large series of human cancer cell lines in vitro and in vivo has been demonstrated for anti-EGFR blocking MAbs or for 8-Cl-cAMP. Therefore, the combination of MAb C225 and 8-Cl-cAMP following chemotherapy could be investigated in cancer patients.

8-Bromo Cyclic Adenosine Monophosphate↗
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

Expression of Wnt5a is downregulated by extracellular matrix and mutated c-Ha-ras in the human mammary epithelial cell line MCF-10A.

Wnt genes are involved in tumour growth and regulate cell adhesion. Some (Wnt5a and Wnt7b) are more highly expressed in human breast cancer compared to normal tissues. Wnt5a is involved in the regulation of cell movement in Xenopus and is upregulated in several human cancers. Factors regulating Wnt gene expression in human breast epithelium are poorly understood, but c-erbB2 is amplified in many breast cancers and associated with rapid growth and metastasis, as is high expression of c-Ha-ras. To further understand the regulation of Wnt gene expression, this study investigated the effect of proto-oncogenes c-Ha-ras and c-erbB2, and collagen on Wnt mRNA expression, in a normal spontaneously immortalised human mammary epithelial cell line MCF-10A. Out of nine human Wnt genes investigated, Wnt5a and Wnt7b were expressed in the parental cell line, and neomycin-, c-Ha-ras- and c-erbB2-transfected cell lines. The level of Wnt5a mRNA expression was decreased 40-fold and 3-fold when parental cells were grown on collagen and in collagen, respectively. This downregulation correlated with cell branching. However, Wnt7b was not regulated by collagen. In the presence of activated c-Ha-ras, the level of Wnt5a mRNA expression was markedly decreased (> 200-fold) and cell growth rate was elevated. When treated with p21ras inhibitor, BZA-5B, there was a moderate reversal of Wnt5a mRNA expression (2-fold) with a parallel decrease in cell growth. The data indicate that c-Ha-ras is an upstream inhibitory regulator of Wnt5a, and provide further evidence of an inverse relationship between Wnt5a mRNA expression and cell branching. This demonstrates selectivity of regulation of individual members of the Wnt gene family by the ras pathway. Overexpression of c-erbB2 had no effect on Wnt5a or Wnt7b mRNA expression. Thus, extracellular matrix and ras regulate Wnt5a, providing a mechanism for feedback of morphogenetic movements, which is relevant also to cancer biology.

Benzodiazepines↗
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

Angiogenesis as a prognostic indicator of survival in non-small-cell lung carcinoma: a prospective study.

BACKGROUND: Tumors acquire nutrients that are essential for continued growth and an avenue for dissemination to the rest of the body by inducing angiogenesis (i.e., the formation of new blood vessels). Preliminary studies involving a number of different kinds of cancer have indicated that an assessment of tumor angiogenesis may be useful in predicting disease outcome. PURPOSE: In a prospective study, we evaluated the relationship between tumor angiogenesis and survival for 407 patients with non-small-cell lung carcinoma who were treated with potentially curative surgery. METHODS: The study population consisted of 360 male and 47 female patients who underwent surgery consecutively at the Department of Surgery, University of Pisa, Italy, from March 1991 through December 1994. Follow-up lasted through February 1996, with a median follow-up for living patients of 29 months (range, 15-60 months). An anti-CD34 monoclonal antibody, which is specific for endothelial cells, and standard immunohistochemical techniques were used to measure angiogenesis in tumor samples. Angiogenesis was quantified in terms of microvessel counts; the counts for single, high-power microscopic fields (magnification x250) in the three most intense areas of blood vessel growth for each sample were averaged. The median microvessel count in this series was 20, and the counts were categorized as follows: 1) low versus high (< or =20 versus >20 microvessels) or 2) in five categories (1-10, 11-20, 21-30, 31-40, and > or =41 microvessels). Disease-free and overall survival during follow-up were assessed. Kaplan-Meier survival curves were modeled in a univariate analysis of patient and tumor characteristics; the Cox proportional hazards model was used in multivariate analysis. Reported P values are two-sided. RESULTS AND CONCLUSIONS: In the univariate analysis, patients with larger tumors (P for trend <.00001), a more advanced tumor stage (P for trend <.00001), a greater degree of regional lymph node involvement (P for trend <.00001), or more vascularized tumors (high versus low microvessel count, P<.00001) experienced significantly reduced overall survival. When microvessel counts were analyzed in five categories, a highly significant trend (P<.00001) toward worse prognosis was observed with increasing tumor vascularity. In multivariate analysis, tumor microvessel count (P<.00001), tumor size (P = .0006), and regional lymph node status (P<.00001) retained independent prognostic value with respect to overall survival; among these variables, tumor microvessel count, considered as a continuous variable, was the most important, with a relative hazard of death of 8.38 (95% confidence interval = 4.19-16.78) associated with the highest microvessel counts. IMPLICATIONS: An evaluation of tumor angiogenesis may be useful in the postsurgical staging of patients with non-small-cell lung carcinoma and in identifying subsets of patients who may benefit from different postsurgical treatments.

Adult↗
PubMed1997

The RIalpha subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-receptor.

Functional interactions between protein kinase A (PKA) and epidermal growth factor receptor (EGF-R) signalling pathways have been suggested. Unlike the type II isoform of PKA (PKAII), the type I (PKAI) and/or its regulatory subunit RIalpha are generally overexpressed in cancer cells and are induced following transforming growth factor alpha (TGF alpha)/EGF-R-dependent transformation. Downregulation of RIalpha/PKAI inhibits TGF alpha expression and EGF-R-dependent signalling. We have previously shown that addition of EGF to quiescent human normal epithelial MCF-10A cells determines PKAI expression and cell membrane translocation before cells enter S phase, while PKAI inhibition prevents S phase entry. Constitutive overexpression of PKAI confers the ability to grow in serum free medium, bypassing EGF requirement. Here we demonstrate a direct interaction of PKAI, but not of PKAII, with the activated EGF-R, that occurs within 5 min following EGF treatment of MCF-10A cells. Moreover, induction of mitogen-activated protein kinase (MAPK) activity following EGF-R activation is mimicked by PKAI overexpression and inhibited by downregulators of PKAI. Finally, the PKAI-EGF-R association occurs through the binding of RIalpha to the SH3 domain(s) of Grb2 adaptor protein, thus allowing the recruitment of the PKAI holoenzyme to the activated EGF-R. This is the first demonstration of a direct interaction of PKAI with the activated EGF-R macromolecular signalling complex.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Cripto enhances the tyrosine phosphorylation of Shc and activates mitogen-activated protein kinase (MAPK) in mammary epithelial cells.

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor alpha, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin beta1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42(erk-2) mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Animals↗
PubMed1997

EGF-related peptides in the pathophysiology of the mammary gland.

Normal mammary gland development is the result of complex interactions between a number of hormones and growth factors. Normal and malignant human mammary epithelial cells are able to synthesize and to respond to various different, locally acting growth factors and growth inhibitors. Among these, the EGF-related peptides play an important role in regulating the proliferation and differentiation of human mammary epithelial cells. EGF4 and TGF4 are able to stimulate the lobulo-alveolar development of the mammary gland in vivo as well they are involved in the pathogenesis of human breast cancer. Experimental evidence suggests that estrogen-induced proliferation of breast carcinoma cells is mediated in part by EGF-related growth factors. It has also been demonstrated that activation of certain cellular protooncogenes such as c-Ha-ras in human mammary epithelial cells results in cellular transformation and in an increased production of several EGF-related growth factors such as TGFalpha and amphiregulin. Coexpression of both EGF-related peptides and their own receptors frequently occurs in human breast carcinomas and in human breast cancer cell lines, suggesting that an autocrine pathway of uncontrolled cell growth sustains neoplastic transformation.

Animals↗
PubMed1997

8-Chloro-cyclic AMP inhibits autocrine and angiogenic growth factor production in human colorectal and breast cancer.

8-Chloro-cyclic AMP (8-Cl-cAMP) is a cAMP analogue that specifically down-regulates type I protein kinase A, a signaling protein directly involved in cell proliferation and neoplastic transformation, and that causes growth inhibition in a variety of human cancer cell types. In this report, we have investigated the effects of 8-Cl-cAMP on the expression of several growth factors in human colon (GEO and LS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines a significant dose- and time-dependent inhibition in the expression of various endogenous autocrine growth factors, such as transforming growth factor alpha, amphiregulin, and CRIPTO, and of two angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor, at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all three cell lines to invade a basement membrane matrix in a chemoinvasion assay. Finally, 8-Cl-cAMP-induced inhibition of GEO tumor growth in nude mice was accompanied by a significant suppression of transforming growth factor alpha, amphiregulin, CRIPTO, basic fibroblast growth factor, and vascular endothelial growth factor production by the tumor cells, and of neoangiogenesis, as detected by factor VIII staining of host blood cells. These results demonstrate that 8-Cl-cAMP is a novel anticancer drug that inhibits the production of various autocrine and paracrine tumor growth factors that are important in sustaining autonomous local growth and facilitate invasion and metastasis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Antitumor activity of combined blockade of epidermal growth factor receptor and protein kinase A.

BACKGROUND: Epidermal growth factor (EGF)-related proteins, such as transforming growth factor-alpha (TGF-alpha), control cancer cell growth through hormonal pathways (i.e., autocrine [hormone acts on cell that produces it] and paracrine [hormone acts on nearby cells] pathways). Overexpression of TGF-alpha and/or its receptor (EGFR) has been detected in human cancers. The blockade of EGFR activation by the use of anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is generally overexpressed in human cancer cells and is involved in neoplastic transformation. Inhibition of PKAI by selective cAMP analogues, such as 8-chloro-cAMP (8-CI-cAMP), induces growth inhibition in various human cancer cell lines. PURPOSE: On the basis of our previous observations of a cooperative anti-proliferative effect of anti-EGFR Mab 528 and 8-Cl-cAMP in human cancer cell lines in vitro, we evaluated the anticancer activity in vivo of the combination of an anti-EGFR MAb (MAb C225) and 8-Cl-cAMP. METHODS: Athymic mice were injected subcutaneously with 10(7) human colon carcinoma GEO cells. After 7 days, when established tumor xenografts of 0.30-0.35 cm3 were detectable, 10-15 mice per group were treated intraperitoneally twice weekly with different doses of 8-Cl-cAMP and/or MAb C225. Cancer cell expression of various growth factors was evaluated by immunohistochemical analysis in tumors obtained from control and treated mice. Data were evaluated for statistical significance using the Student's t test and the Mantel-Cox logrank test. All P values represent two-sided tests of statistical significance. RESULTS: A 5-week treatment with low doses of 8-Cl-cAMP (0.5 mg/dose) and MAb C225 (0.25 mg/dose) blocked GEO tumor growth (compared with that in control mice; P < .00001) and suppressed cancer cell production of autocrine growth factors, such as TGF-alpha, amphiregulin, and CRIPTO, and of angiogenic (promotes new blood vessel formation) factors, such as vascular endothelial growth factor and basic fibroblast growth factor, with no signs of toxicity. Control and 8-Cl-cAMP (0.5 mg/dose)-treated mice died within 9-10 weeks after tumor cell injection. In MAb C225 (0.25 mg/dose)-treated mice, GEO tumors resumed a growth rate comparable to that in control animals within 3 weeks following the end of treatment and the mice died between 11 and 20 weeks after tumor cell injection. GEO tumor growth was significantly delayed in the MAb C225 plus 8-Cl-cAMP treatment group (P < .00001) and was accompanied by a prolonged survival of mice (P < .00001) as compared with the control group. CONCLUSIONS: Long-term treatment with a combination of agents that selectively inhibit two intracellular signal-transduction enzymes, such as the PKAI serine-threonine kinase and the EGFR tyrosine kinase, has anticancer activity in vivo, reflected by suppression of tumor proliferation and angiogenesis, with no signs of toxicity. IMPLICATIONS: Since these inhibitors of intracellular mitogenic (growth-stimulating) signaling have a different mechanism(s) of action and do not antagonize the effects of cytotoxic therapy, a combination of anti-EGFR MAb C225 and 8-Cl-cAMP should be investigated as a nontoxic, long-term treatment for cancer patients following chemotherapy.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Transfection with a CRIPTO anti-sense plasmid suppresses endogenous CRIPTO expression and inhibits transformation in a human embryonal carcinoma cell line.

CRIPTO is a member of the epidermal growth factor (EGF) gene family originally isolated from undifferentiated human NTERA2 clone D1 (NT2D1) multipotent embryonal carcinoma cells. Retinoic acid (RA) treatment of NT2D1 cells leads to a neuronal differentiation program and to concomitant loss of CRIPTO mRNA expression. To assess the role of CRIPTO in the control of NT2D1 cell growth or differentiation, these cells were treated with 3 anti-sense oligodeoxynucleotides complementary to the 5' end of the human CRIPTO mRNA. A dose-dependent inhibition of monolayer and soft agar growth was observed with each of these CRIPTO anti-sense oligodeoxynucleotides but not with a control oligodeoxynucleotide of random sequence or with the 3 corresponding CRIPTO sense oligodeoxynucleotides. In addition, NT2D1 cells were transfected with a recombinant expression vector containing a 918-bp coding fragment of the human CRIPTO cDNA in the 3' to 5' orientation. NT2D1 CRIPTO anti-sense transfectants exhibited a significantly reduced endogenous CRIPTO mRNA and protein, a 4- to 5-fold decrease in growth rate in monolayer and a 50-70% reduction in cloning efficiency in soft agar as compared with NT2D1 parental cells or with NT2D1 cells transfected with a plasmid containing the neomycin-resistance gene alone (NT2D1 neo cells). Finally, we examined the expression of immunophenotypic markers that are modulated during the differentiation of NT2D1 cells following RA treatment. The globoseries stage-specific embryonic antigen-3 recognized by the monoclonal antibody (MAb) SSEA-3 was expressed in 60% of undifferentiated parental NT2D1 or NT2D1 neo cells and in only 20% of NT2D1 CRIPTO anti-sense transfectants, whereas it was down-regulated in all cell lines following RA treatment. A neuroectodermal antigen recognized by the A2B5 MAb, which was not expressed in parental NT2D1, in NT2D1 neo or in CRIPTO anti-sense NT2D1 cells, was induced by RA treatment in all cell lines. Taken together, our results show that inhibition of endogenous CRIPTO expression in human embryonal carcinoma cells interferes with both transformation and differentiation.

Base Sequence↗
PubMed1996

Enhanced expression of heregulin in c-erb B-2 and c-Ha-ras transformed mouse and human mammary epithelial cells.

Heregulin beta 1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor alpha, or amphiregulin, heregulin beta 1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-independent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin beta 1, whereas heregulin beta 1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells.

Animals↗
PubMed1996

Differential immunohistochemical detection of transforming growth factor alpha, amphiregulin and CRIPTO in human normal and malignant breast tissues.

The expression of growth factors, such as transforming growth factor alpha (TGF alpha), amphiregulin (AR) and CRIPTO, a type-1 tyrosine-kinase growth factor receptor (erbB-2), and a tumor-suppressor gene (p53), that have been implicated in the development and/or the progression of breast cancer, was evaluated by immunohistochemistry in 100 human primary infiltrating breast carcinomas (IBC). AR and CRIPTO immunoreactivity was also assessed in 55 human breast ductal carcinomas in situ (DCIS). Within the 100 IBC, 80, 50, 73, 17, and 34 tumors expressed moderate to high levels of TGF alpha, AR, CRIPTO, erbB-2, and p53 respectively. In addition, AR and CRIPTO immunoreactivity were found in 11 and in 26 out of 55 DCIS respectively. In contrast, only 4, 3, and 2 out of 10 normal mammary-gland samples were weakly positive for TGF alpha, AR, and CRIPTO expression, respectively, whereas none was positive for erbB-2 or p53. Within the 100 IBC, expression of erbB-2 significantly correlated with high histologic and nuclear grading, with high growth fraction, and with estrogen-receptor (ER)- and progesterone-receptor (PgR)-negative tumors. A statistically significant correlation was also observed between p53 expression and high histologic grading, high growth fraction, and PgR-negative tumors. In contrast, no significant correlations were found between TGF alpha, AR, and CRIPTO immunoreactivity and various clinicopathological parameters, with the exception of a positive correlation between TGF alpha and ER expression. These data demonstrate that TGF alpha, AR, and CRIPTO expression are significantly increased in malignant mammary epithelium relative to normal epithelium. In particular, the differential expression of CRIPTO may serve as a potential tumor marker for breast carcinogenesis.

Adult↗