Biomedical subjects

Carlo M Croce

Publications and source records attributed to Carlo M Croce.

At least 91 records · Page 5Linked to original sources

PubMed2003

Regression of upper gastric cancer in mice by FHIT gene delivery.

Fhit expression is reduced in most cancers, and Fhit replacement by FHIT expression viruses in lung, esophageal, pancreatic, and cervical cancers induces apoptosis in the cancer cells. Mice carrying one or two inactivated Fhit alleles are hypersensitive to development of N-nitrosomethylbenzylamine (NMBA)-induced forestomach tumors. In the present study, we investigated the kinetics and mechanism of tumor reversal and intervention by oral delivery of FHIT expression viruses. Tumor analysis showed that: a) by 37 days post-NMBA, control mice showed approximately 7 tumors and by 84 days approximately 10 tumors/forestomach; b) mice receiving FHIT virus at 2 or 42 days post-NMBA showed significantly reduced tumor burdens; c) Fhit was still expressed at 82 days postinfection; d) control viral infection had no effect on tumor development; and e) reduced Bcl2, increased Bax expression, and increased TUNEL-positive apoptotic nuclei characterized the restored epithelia of FHIT transduced forestomachs. Thus, FHIT viral gene delivery prevents or retards development of carcinogen-induced forestomach tumors and reverses development of established tumors by 60-70% through an apoptotic pathway. This dramatic reduction in tumor burden emphasizes the efficacy of targeting the FHIT apoptotic pathway for tumor eradication.

Acid Anhydride Hydrolases↗

PubMed2003

Allele loss and promoter hypermethylation of VHL, RAR-beta, RASSF1A, and FHIT tumor suppressor genes on chromosome 3p in esophageal squamous cell carcinoma.

Promoter hypermethylation is an alternative way to inactivate tumor suppressor genes in cancer. Alterations of chromosome 3p are frequently involved in many types of cancer, including esophageal squamous cell carcinoma. Here, we investigated the methylation status and loss of heterozygosity (LOH) of 3p tumor suppressor genes. We examined the promoter methylation status of von Hippel-Lindau disease (VHL), retinoic acid receptor beta (RAR-beta), RAS association domain family 1A (RASSF1A), and fragile histidine triad (FHIT) genes in 22 esophageal squamous cell carcinoma cell lines and 47 primary tumors and corresponding noncancerous tissues by a methylation-specific PCR. In addition, we analyzed 47 paired samples for LOH at eight loci on chromosome 3p. Hypermethylation in VHL, RAR-beta, RASSF1A, and FHIT was detected in 36, 73, 73, and 50% of tumor cell lines, respectively. In primary tumors, hypermethylation in VHL, RAR-beta, RASSF1A, and FHIT was detected in 13, 55, 51, and 45%, respectively. In corresponding noncancerous tissues, hypermethylation in RAR-beta and FHIT was frequently detected in 38 and 30%, respectively, whereas no VHL hypermethylation and only 4% of RASSF1A hypermethylation were detected. Furthermore, in clinical stages I and II, hypermethylation in RAR-beta (67%) and FHIT (78%) was frequently detected, whereas no VHL hypermethylation and 11% of RASSF1A hypermethylation were detected. On the other hand, the correlation between FHIT hypermethylation and LOH at FHIT region was statistically significant (P = 0.008). Our findings suggest that hypermethylation of the RAR-beta and FHIT may play an important role in the early stage of esophageal squamous cell carcinogenesis. In addition, FHIT may be inactivated in accordance with the two-hit inactivation model, involving deletion of one allele and hypermethylation of the other.

Acid Anhydride Hydrolases↗

PubMed2003

Loss of Hmga1 gene function affects embryonic stem cell lympho-hematopoietic differentiation.

By interacting with transcription machinery, high-mobility group A 1 (HMGA1) proteins alter the chromatin structure and thereby regulate the transcriptional activity of several genes. To assess their role in development, we studied the in vitro differentiation of embryonic stem (ES) cells that bear one or both disrupted Hmga1 alleles. Here, we report that Hmga1 null ES cells generate fewer T-cell precursors than do wild-type ES cells. Indeed, they preferentially differentiate to B cells, probably consequent to decreased interleukin 2 expression and increased interleukin 6 expression. Moreover, a lack of HMGA1 expression induces changes in hemopoietic differentiation, i.e., a reduced monocyte/macrophage population and an increase in megakaryocyte precursor numbers, erythropoiesis, and globin gene expression. Re-expression of the Hmga1 gene in Hmga1 null ES cells restores the wild-type phenotype. The effect on megakaryocyte/erythrocyte lineages seems, at least in part, mediated by the GATA-1 transcription factor, a key regulator of red blood cell differentiation. In fact, we found that Hmga1-/- ES cells overexpress GATA-1 and that HMGA1 proteins directly control GATA-1 transcription. Taken together, these data indicate that HMGA1 proteins play a prime role in lymphohematopoietic differentiation.

Animals↗

PubMed2003

Allelic loss on chromosome 3p21.3 and promoter hypermethylation of semaphorin 3B in non-small cell lung cancer.

The aim of this study was to evaluate the promoter methylation status and loss of heterozygosity (LOH) of the SEMA3B in non-small cell lung cancers (NSCLCs). We analyzed the methylation status of semaphorin 3B (SEMA3B) promoter and LOH at 3p21.3 in eight NSCLC cell lines and 27 primary tumors. Hypermethylation of SEMA3B was found in 50% of the cell lines and 41% of the primary tumors studied. The presence of hypermethylation was statistically associated with loss of SEMA3B expression in both cell lines (P = 0.02) and primary tumors (P < 0.01). There was no correlation between SEMA3B and tumor stage. On the other hand, the correlation between SEMA3B methylation status and LOH at 3p21.3 was significant (P = 0.02). Notably, 7 of 8 tumors with both hypermethylation and LOH of SEMA3B showed the absence of the expression. Treatment with 5-AZAC restored SEMA3B expression in NSCLC cell line. These results indicate that SEMA3B gene alterations may play a important role in the malignant transformation of NSCLC via a two-hit mechanism, including epigenetic changes and allelic loss, for tumor suppressor gene inactivation.

Aged↗

PubMed2003

Parkin, a gene implicated in autosomal recessive juvenile parkinsonism, is a candidate tumor suppressor gene on chromosome 6q25-q27.

In an effort to identify tumor suppressor gene(s) associated with the frequent loss of heterozygosity observed on chromosome 6q25-q27, we constructed a contig derived from the sequences of bacterial artificial chromosomeP1 bacteriophage artificial chromosome clones defined by the genetic interval D6S1581-D6S1579-D6S305-D6S1599-D6S1008. Sequence analysis of this contig found it to contain eight known genes, including the complete genomic structure of the Parkin gene. Loss of heterozygosity (LOH) analysis of 40 malignant breast and ovarian tumors identified a common minimal region of loss, including the markers D6S305 (50%) and D6S1599 (32%). Both loci exhibited the highest frequencies of LOH in this study and are each located within the Parkin genomic structure. Whereas mutation analysis revealed no missense substitutions, expression of the Parkin gene appeared to be down-regulated or absent in the tumor biopsies and tumor cell lines examined. In addition, the identification of two truncating deletions in 3 of 20 ovarian tumor samples, as well as homozygous deletion of exon 2 in the lung adenocarcinoma cell lines Calu-3 and H-1573, supports the hypothesis that hemizygous or homozygous deletions are responsible for the abnormal expression of Parkin in these samples. These data suggest that the LOH observed at chromosome 6q25-q26 may contribute to the initiation andor progression of cancer by inactivating or reducing the expression of the Parkin gene. Because Parkin maps to FRA6E, one of the most active common fragile sites in the human genome, it represents another example of a large tumor suppressor gene, like FHIT and WWOX, located at a common fragile site.

Base Sequence↗

PubMed2003

Restoration of fragile histidine triad (FHIT) expression induces apoptosis and suppresses tumorigenicity in breast cancer cell lines.

The fragile histidine triad (FHIT) gene at chromosome 3p14.2 is a tumor suppressor gene that is altered mainly by deletion in a large fraction of human tumors, including breast cancers. To evaluate the potential of FHIT gene therapy in this type of cancer, we have studied the biological effects of adenoviral FHIT transduction (Ad-FHIT) in breast cancer cell lines. The results showed that, after FHIT restoration in BT-549, MDA-MB-436, and HCC1806 cells, they underwent apoptosis by activation of the intrinsic pathway. In all three cell lines infected with Ad-FHIT, we have found activation of caspase-2, which is required for permeabilization of mitochondria, release of cytochrome c, and apoptosis. Furthermore, Fhit overexpression produces alteration in cell cycling properties, as well as reduction of the tumorigenic potential in nude mice.

Acid Anhydride Hydrolases↗

PubMed2003

WW domain containing oxidoreductase gene expression is altered in non-small cell lung cancer.

WWOX (WW domain containing oxidoreductase), a putative tumor suppressor gene that maps to the common fragile site FRA16D on chromosome 16q23.3-24.1, is altered in breast, esophageal, and ovarian cancer. Because the FRA3B/FHIT locus at 3p14.2 is a preferential target for genetic changes caused by tobacco smoke, we intended to evaluate the status of the FRA16D/WWOX gene in non-small cell lung cancer; we have analyzed 27 paired normal and tumor lung tissues and 8 lung cancer cell lines for WWOX alterations by reverse transcriptase-PCR, loss of heterozygosity, and mutation analysis. Transcripts missing WWOX exons were detected in 7 primary tumors (7 of 27; 25.9%) and 5 of 8 cell lines. In addition, loss of heterozygosity at the WWOX locus was observed in 10 primary tumors (10 of 27; 37.0%). We conclude that WWOX alterations occur in a significant fraction of lung cancers and may contribute to the pathogenesis of non-small cell lung cancer.

Carcinoma, Non-Small-Cell Lung↗

PubMed2003

Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding.

The FHIT gene is inactivated early in the development of many human tumors, and Fhit-deficient mice have increased cancer incidence. Viral reexpression of Fhit kills Fhit-deficient cells by induction of apoptosis. Fhit, a member of branch 2 of the histidine-triad superfamily of nucleoside monophosphate hydrolases and transferases, is a diadenosine polyphosphate hydrolase, the active-site histidine of which is not required for tumor suppression. To provide a rigorous test of the hypothesis that Fhit function depends on forming a complex with substrates, we designed a series of alleles of Fhit intended to reduce substrate-binding andor hydrolytic rates, characterized these mutants biochemically, and then performed quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in k(cat) and increases as large as 30-fold in K(M). Nonetheless, when mutant FHIT genes were expressed in two human cancer cell lines containing FHIT deletions, reductions in apoptotic activity correlated exclusively with K(M). Mutants with 2- and 7-fold increases in K(M) significantly reduced apoptotic indices, whereas the mutant with a 30-fold increase in K(M) retained little cellular function. These data indicate that the proapoptotic function of Fhit is limited by substrate binding and is unrelated to substrate hydrolysis.

Acid Anhydride Hydrolases↗

PubMed2003

p53 deficiency accelerates induction and progression of esophageal and forestomach tumors in zinc-deficient mice.

The p53 tumor suppressor protein plays a pivotal role in preventing uncontrolled cellular proliferation. By contrast, zinc deprivation enhances esophageal cell proliferation and the induction of esophageal tumors in rodents by N-nitrosomethylbenzylamine (NMBA). We investigated whether p53 deficiency rendered zinc-deficient (ZD) mice more susceptible to NMBA-induced esophageal/forestomach carcinogenesis. At 6-7 weeks of age, p53 null (-/-), heterozygous (+/-), and wild-type (+/+) mice were placed on ZD or zinc-sufficient (ZS) diets to form six experimental groups: ZD:p53-/-; ZD:p53+/-; ZD:p53+/+; ZS:p53-/-; ZS:p53+/-; and ZS:p53+/+. After 3 weeks, 15-23 mice in each group were treated once with NMBA (2 mg/kg body weight). Control animals were untreated. Zinc deficiency alone induced unrestrained cellular proliferation in the esophagus and forestomach of p53-/- mice. Forestomach tumors were first detected in a ZD:p53-/- mouse at 13 days. By 30 days, 100% (21 of 21) of ZD:p53-/- mice developed forestomach tumors and 38% showed esophageal tumors versus 42 and 0% in ZS:p53-/- mice (P < 0.004, esophagus; P < 0.001, forestomach). ZD:p53-/- mice showed an accelerated progression to malignancy, with 10% of esophageal tumors and 38% of forestomach tumors presenting as carcinomas. Nearly 20% of ZD:p53-/- mice developed esophageal Barrett's metaplasia, a lesion not previously seen in NMBA-induced neoplasia. ZD:p53+/- mice had significantly higher tumor incidence than ZS:p53+/- mice. The order of tumor incidence in forestomach was as follows: naught incidence in ZS:p53+/+ mice; ZD:p53-/- > ZD:p53+/- > ZS:p53-/- > ZD:p53+/+ >/= ZS:p53+/- > ZS:p53+/+. The rapid rate of tumor induction/progression in ZD:p53-/- mice was accompanied by an increase in the rate of cell proliferation and a decrease in apoptosis. cDNA array expression analysis of known genes identified a 5-fold up-regulation of cytokeratin 14 mRNA expression in ZD:p53-/- versus ZS:p53-/- forestomach, a result showing gene-modulating effects of zinc deficiency. Cytokeratin 14 is a biomarker in human esophageal carcinogenesis. Our findings provide in vivo evidence for the collaboration of a deficiency of both p53 and zinc in esophageal carcinogenesis and reveal molecular targets of this collaboration.

Animals↗

PubMed2003

Regulation of BRCA1 transcription by specific single-stranded DNA binding factors.

Since the majority of high-grade breast cancers express reduced levels of BRCA1 mRNA, we investigated the factors regulating BRCA1 transcription. Factors with specific affinity for the previously identified positive regulatory region (PRR) in the BRCA1 promoter were purified from whole-cell extracts. Identified proteins included replication protein A and a series of related factors with affinity for the sense strand of PRR. A subset of the identified factors activated the BRCA1 promoter. Identification of these families of proteins regulating the BRCA1 promoter represents an important step in the comprehension of the mechanisms responsible for breast cancer development.

Base Sequence↗

PubMed2003

Negative regulation of BRCA1 gene expression by HMGA1 proteins accounts for the reduced BRCA1 protein levels in sporadic breast carcinoma.

A drastic reduction in BRCA1 gene expression is a characteristic feature of aggressive sporadic breast carcinoma. However, the mechanisms underlying BRCA1 downregulation in breast cancer are not well understood. Here we report that both in vitro and in vivo HMGA1b protein binds to and inhibits the activity of both human and mouse BRCA1 promoters. Consistently, murine embryonic stem (ES) cells with the Hmga1 gene deleted display higher Brca1 mRNA and protein levels than do wild-type ES cells. Stable transfection of MCF-7 cells with the HMGA1b cDNA results in a decrease of BRCA1 gene expression and in a lack of BRCA1 induction after estrogen treatment. Finally, we found an inverse correlation between HMGA1 and BRCA1 mRNA and protein expression in human mammary carcinoma cell lines and tissues. These data indicate that HMGA1 proteins are involved in transcriptional regulation of the BRCA1 gene, and their overexpression may have a role in BRCA1 downregulation observed in aggressive mammary carcinomas.

Animals↗

PubMed2003

DLX genes as targets of ALL-1: DLX 2,3,4 down-regulation in t(4;11) acute lymphoblastic leukemias.

Dlx genes constitute a gene family thought to be essential in morphogenesis and development. We show here that in vertebrate cells, Dlx genes appear to be part of a regulatory cascade initiated by acute lymphoblastic leukemia (ALL)-1, a master regulator gene whose disruption is implicated in several human acute leukemias. The expression of Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7 was absent in All-1 -/- mouse embryonic stem cells and reduced in All-1 +/- cells. In leukemic patients affected by the t(4;11)(q21;q23) chromosomal abnormality, the expression of DLX2, DLX3, and DLX4 was virtually abrogated. Our data indicate that Dlx genes are downstream targets of ALL-1 and could be considered as important tools for the study of the early leukemic cell phenotype.

Animals↗

PubMed2003

Targeting mature T cell leukemia: new understanding of molecular pathways.

The best studied T cell leukemia/lymphoma from a genetic and biochemical point of view is T-cell chronic lymphocytic/prolymphocytic leukemia (T-CLL/T-PLL). This neoplasia commonly shows chromosomal rearrangements at 14q32.1 including translocations [t(14;14)(q11;q32), t(7;14)(q35;q32)], and inversions [inv(14)(q11;q32)]. The investigation of the locus in question at 14q32.1 resulted in the identification of two related genes named T cell leukemia/lymphoma 1 (TCL1) and TCL1b. Both genes are activated in T-CLL/T-PLL by the chromosomal aberrations mentioned above. Mice from a transgenic mouse strain expressing the TCL1 gene under the thymocyte specific lck promoter developed a mature T cell leukemia late in life, thereby demonstrating that over-expression of TCL1 induces the neoplastic transformation of T cells. Biochemically, Tcl1 protein works as a co-factor of the Akt kinase, a key regulator of antiapoptotic and proliferative signals. Tcl1 interacts physically with Akt, increases its kinase activity and facilitates its transport to the nucleus. The pathogenesis of T-CLL/T-PLL may also involve Nur77, a T cell transcription factor required for T cell receptor-mediated apoptosis. Akt phosphorylates Nur77, thereby blocking its DNA-binding ability and rendering the transcription factor inactive. The recently emerged insights into the molecular mechanisms of T cell leukemogenesis will allow for the development of specific pharmacological tools for the treatment of these hematopoietic malignancies.

Animals↗

PubMed2003

Promoter hypermethylation of RASSF1A in esophageal squamous cell carcinoma.

PURPOSE: The RAS association domain family 1A (RASSF1A) gene, a candidate tumor suppressor gene, is frequently inactivated by hypermethylation of its promoter region in several human cancers. The aim of this study was to evaluate the promoter methylation status of the RASSF1A in esophageal squamous cell carcinoma. EXPERIMENTAL DESIGN: We analyzed the methylation status of RASSF1A promoter by methylation-specific PCR in 23 esophageal squamous cell carcinoma cell lines and 48 primary tumors. RESULTS: Hypermethylation of RASSF1A was found in 74% of cell lines and 52% of primary tumors. The presence of hypermethylation was statistically associated with loss of RASSF1A mRNA expression in both cell lines (P = 0.007) and primary tumors (P = 0.003). There was a statistically significant correlation between the presence of hypermethylation and tumor stage (P = 0.009). CONCLUSIONS: Our findings suggest that epigenetic silencing of RASSF1A gene expression by promoter hypermethylation could play an important role in primary esophageal squamous cell carcinogenesis.

Aged↗

PubMed2003

Expression of FRA16D/WWOX and FRA3B/FHIT genes in hematopoietic malignancies.

The WW domain containing oxidoreductase (WWOX) gene was recently identified as a candidate tumor suppressor gene at a common fragile site, FRA16D. Because the fragile histidine triad (FHIT) gene, a tumor suppressor gene encompassing the most active, common fragile site FRA3B, is frequently deleted in various cancers, we evaluated the expression of WWOX and FHIT in 74 cases of primary hematopoietic neoplasias and 20 leukemia cell lines. Aberration or absence of WWOX transcripts was detected in 51% of the primary cases and 55% of cell lines, and three WWOX nucleotide variants were detected among the leukemia cell lines. FHIT expression was absent or altered in 36% of the primary cases and 15% of cell lines. The occurrence of aberrant FHIT reverse transcription-PCR products correlated significantly with the occurrence of WWOX alterations. Wild-type transcripts of both genes were expressed in normal hematopoiesis along with a small fraction of short transcripts. A DNA blot study showed that WWOX and FHIT genes were deleted in 2 of 18 cases with primary acute leukemias; both genes were not expressed in the 2 cases. Furthermore, treatment of cells with a demethylating or histone acetylating agent in culture resulted in increased expression of WWOX and FHIT mRNA in leukemia cells. Conclusions are that WWOX expression is frequently altered or absent in hematopoietic disorders, often in association with FHIT alterations, and that alterations of these fragile genes may result not only from genomic deletions but also from epigenetic modifications associated with expression of fragility.

Acetylation↗

PubMed2002

Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia.

Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue-specific gene regulation. MiRs are transcribed as short hairpin precursors ( approximately 70 nt) and are processed into active 21- to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via base-pairing interactions. Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL). Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority ( approximately 68%) of CLL cases.

Adult↗

PubMed2002

Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice.

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of beta-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat alpha-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular (dP/dt(max)) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular dP/dt(min), was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, beta-adrenergic receptor (beta-AR) density, and beta-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-beta is phosphorylated, whereas p42/44 mitogen-activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-beta/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting beta-AR signaling capacity.

Animals↗

PubMed2002

Isolation and characterization of a novel gene, hRFI, preferentially expressed in esophageal cancer.

hTID1, a human homologue of Drosophila tumor suppressor, I(2)tid regulates the release of cytochrome c from mitochondria and subsequent alteration of caspase-3 activity on apoptosis induced by exogenous stimuli, such as tumor necrosis factor-alpha and mitomycin C. To search for an interacting molecule with hTid1, we applied two-hybrid yeast screening and isolated a novel gene, which encodes a 46 kDa protein of 373 residues. Within the deduced amino acid sequence, a region showing homology to the Ring Finger domain of X-linked inhibitor of apoptosis protein was identified and the gene was designated as hRFI, standing for human Ring Finger homologous to IAP type. A 2.0 kb hRFI transcript was ubiquitously expressed in all human tissues as well as several cancer cell lines examined. Northern blot analysis showed that in 70% (14 out of 20) of esophageal cancer patients, expression of hRFI in cancerous regions was two or more times higher than in the corresponding normal tissues. HeLa cells transfected with hRFI construct exhibited a tendency to resist TNF-alpha induced apoptosis, suggesting an anti-apoptotic function of the hRFI product. Finally, hRFI protein was shown to be cleaved within the DEDD sequence spanning residues 230-233 by caspase-3 during the apoptotic induction.

Amino Acid Sequence↗