Showing papers by "Carlo M. Croce published in 1996"

Taxol induces bcl-2 phosphorylation and death of prostate cancer cells.

Abstract: Treatment of prostate cancer cell lines expressing bcl-2 with taxol induces bcl-2 phosphorylation and programmed cell death, whereas treatment of bcl-2-negative prostate cancer cells with taxol does not induce apoptosis. bcl-2 phosphorylation seems to inhibit its binding to bax since less bax was observed in immunocomplex with bcl-2 in taxol-treated cancer cells. These findings support the use of the anticancer drug taxol for the treatment of bcl-2-positive prostate cancers and other bcl-2-positive malignancies, such as follicular lymphoma.

FHIT gene alterations in head and neck squamous cell carcinomas

Abstract: To determine whether the FHIT gene at 3p14.2 is altered in head and neck squamous cell carcinomas (HNSCC), we examined 26 HNSCC cell lines for deletions within the FHIT locus by Southern analysis, for allelic losses of specific exons FHIT by fluorescence in situ hybridization (FISH) and for integrity of FHIT transcripts. Three cell lines exhibited homozygous deletions within the FHIT gene, 55% (15/25) showed the presence of aberrant transcripts, and 65% (13/20) showed the presence of multiple cell populations with losses of different portions of FHIT alleles by FISH of FHIT genomic clones to interphase nuclei. When the data obtained by FISH and by reverse transcriptase-PCR analyses are combined, 22 of 26 cell lines showed alterations of at least one allele of the FHIT gene. Our data indicate that the FHIT gene is disrupted in HNSCCs and hence, loss of FHIT function may be important in the development and/or progression of head and neck cancers.

The FHIT Gene at 3p14.2 Is Abnormal in Breast Carcinomas

Abstract: Chromosome 3p deletions in breast cancer have been detected at 3p12–p21 by cytogenetic and loss of heterozygosity studies. Recently, we have cloned the FHIT (fragile histidine triad) gene, located at 3p14.2. Abnormalities of the FHIT locus were found in many established cancer cell lines, and the gene was abnormally transcribed in primary tumors of the digestive tract and lung. In this report, we describe the analysis of breast cancer, cell lines, and primary tumors for alterations in transcription of the FHIT gene; about 20% of the samples exhibited altered transcripts. In most of the cases, aberrant transcripts were missing exons. Lack of expression of FHIT mRNA was observed in another 10% of primary tumor samples. These results suggest that alterations in the FHIT gene may play an important role in breast cancer tumorigenesis and suggest that the FHIT gene product functions in the control of the tumorigenic phenotype in a large variety of human neoplasms.

Partial Tandem Duplication of ALL1 as a Recurrent Molecular Defect in Acute Myeloid Leukemia with Trisomy 11

Abstract: Gains of a single chromosome are frequent cytogenic findings in human cancer, but no molecular rearrangement has been consistently associated with any trisomy. In acute myeloid leukemia (AML), trisomy 11 (+11) occurring as a sole abnormality is the third most common trisomy. We have shown that the ALL1 gene, located at 11q23, can be rearranged as a result of a partial tandem duplication in two such cases of AML. To test the hypothesis that the partial tandem duplication of ALL1 is the recurrent molecular defect in cases of AML presenting with +11 as a sole cytogenic abnormality, we performed Southern analysis and PCR for defects of ALL1 in 17 cases of AML and one case of myelodysplastic syndrome with +11 or +11q but without cytogenic evidence of a structural abnormality involving 11q23. Twelve cases (67%) had rearrangement of ALL1, including 10 of 11 patients (91%) with +11 as a sole abnormality and 2 of 7 cases (29%) with +11 and other aberrations; all were classified as FAB M1 or M2. In 10 of the 12 cases, material was available for additional characterization; a partial tandem duplication of ALL1 was detected in each of these 10 cases (100%). Four cases demonstrated previously unreported duplications, two of which were detectable only by reverse transcription-PCR. Four patients with the ALL1 duplication also displayed a loss of material from 7q, suggesting an association between these two findings. We conclude that the partial tandem duplication of ALL1 is present in most, if not all, cases of AML with +11 as a sole abnormality, and can be found in cases of AML with +11 or +11q accompanied by other cytogenic abnormalities. The duplication is more prevalent in AML than was recognized previously in part because its size and location vary considerably, requiring a variety of molecular probes for detection. Our finding of the ALL1 duplication as a consistent defect in patients with +11 represents the first identification of a specific gene rearrangement associated with recurrent trisomy in human cancer.

Potential gastrointestinal tumor suppressor locus at the 3p14.2 FRA3B site identified by homozygous deletions in tumor cell lines.

Abstract: A number of DNA fragments, identified by representational difference analysis, which were homozygously deleted in various cancer cell lines were previously mapped to human chromosomal arms. One of these, BE758-6, which was homozygously deleted in a number of colon carcinoma cell lines, had been mapped to chromosome region 3p. We have further localized the probe to 3p14.2, ∼350 kbp telomeric to the 3p14.2 break of the t(3;8) hereditary renal cell carcinoma chromosome translocation, within or near the 3p14.2 FRA3B, the most common human fragile site. We determined the sizes of the homozygous deletions in a number of cancer cell lines after isolation of a yeast artificial chromosome contig and development of STS markers which fall between D3S1234 and D3S1481 , which flank the deletions. Homozygous deletions were observed and sized not only in the cell lines originally reported but also in a number of nasopharyngeal carcinoma cell lines and a gastric carcinoma cell line. About 50% of uncultured stomach and colon carcinomas were then shown to lose heterozygosity for alleles in the same region, with a common region of loss between the D3S1234 and D3S1481 markers. Thus, it is likely that the homozygous deletion observed in these cancer cell lines harbors an important tumor suppressor gene for several tumor types.

Prostate specific antigen in breast cancer, benign breast disease and normal breast tissue

Abstract: Prostate specific antigen (PSA) is a tumor marker used widely for the diagnosis and monitoring of prostatic adenocarcinoma. Recently, we provided evidence that PSA may also be produced by breast tumors. In this report we examined quantitatively the PSA levels in 199 breast tumors, 48 tissues with benign breast disease (BBD, 34 fibroadenomas), and 36 normal breast tissues. Significant amounts of PSA (≥ 0.030 ng of PSA per mg of total protein) were found in 28% of breast tumors, 65% of BBD tissues, and 33% of normal breast tissues. PSA positivity in breast tumors was highest in stage I disease (34%) and decreased with disease stage (24% in stage II and 18% in stage III–IV). Using polymerase chain reaction amplification we have shown PSA mRNA presence in patients with PSA protein-positive tissues (benign and malignant) but not in patients with PSA protein-negative tissues. Our data suggest that PSA is expressed frequently by normal breast tissue, by tissue of benign breast diseases, and by breast cancer tissue. Highest expression is seen in benign breast disease and lowest expression in advanced stage cancerous tissue. As PSA production is mediated by steroid hormones and their receptors, we propose that PSA may be a new marker of steroid hormone action in the normal or diseased female breast. The role of this enzyme in the development of breast diseases including breast cancer is currently unknown.

T-Cell-directed TAL-1 Expression Induces T-Cell Malignancies in Transgenic Mice

Abstract: The TAL-1 gene specifies for a basic domain-helix-loop-helix protein, which is involved in the control of normal hematopoiesis. In human pathology, the TAL-1 gene product is expressed in a high percentage of T-cell acute lymphoblastic leukemias in the pediatric age range; however, it has not been established whether the expression has a causal role in oncogenesis. In this report, we describe the phenotype of mouse transgenic lines obtained by inducing tal-1 protein expression in lymphoid tissues using the LCK promoter. The survival rate of tal-1 transgenic animals was much lower as compared with control mice. Histopathological analysis revealed lymphomas of T-cell type, often comprising a minor B-cell component. Some mice showed marked splenic lymphocyte depletion. Primary lymphocyte cultures showed partial independence from exogenous growth stimuli and increased resistance to low-serum apoptosis. To further unravel the tal-1 oncogenic potential, a strain of tal-1 transgenic mice was crossbred with p53-/- mice; the survival rate in these animals was reduced by more than one-half when compared with that of tal-1 mice, and histopathological analysis revealed exclusively T-cell lymphomas. These data indicate that TAL-1, expressed in T cells, is per se a potent oncogene, which may exert a key leukemogenetic role in the majority of T-cell acute lymphoblastic leukemias.

Loss of Heterozygosity for Chromosome 11 in Adenocarcinoma of the Stomach

Abstract: Loss of heterozygosity (LOH) at several chromosomal loci is a common feature of the malignant progression of human tumors. In the case of chromosome 11, LOH has been well documented in several types of solid neoplasms, including gastric carcinoma, suggesting the presence of suppressor gene(s) at 11p15 and 11q22–23. Little is currently known about the molecular events occurring during the development of gastric cancer. To define the regions of chromosome 11 involved in gastric cancer progression, we used high-density polymorphic markers to screen for LOH in matched normal and tumor tissue DNA from 60 primary gastric carcinomas. We found that 21% of the tumors showed LOH simultaneously at 11p15 and 11q22–23, 41% had LOH at 11p15, and 30% had LOH at 11q22–23. We confirm that the minimal critical area of LOH for 11p15.5 is the approximately 2-Mb region between loci D11S1318 and D11S988 . However, when we analyzed the pattern of LOH according to the country of origin of the patient, LOH for 11q22–23 alone was found only in cases from Italy. The minimal critical region of LOH at 11q22–23 is identical to that identified for other solid tumors, suggesting that the same putative tumor suppressor gene(s) contained within this region is involved in the pathogenesis of several common human tumors.

Double Knockout of the ALL-1 Gene Blocks Hematopoietic Differentiation in Vitro

Abstract: The ALL-1 gene is involved in translocations with many partner genes in different types of acute leukemias, but it is not clear whether it acts as an oncogene or whether the fusion proteins resulting from the translocations have dominant negative effects. To distinguish between these two possibilities, we analyzed the ability of wild-type AB2.1 embryonal stem (ES) cells and of single or double ALL-1 gene knockout cells derived from them to differentiate along hematopoietic lineages after withdrawal of leukemia inhibitory factor, using in vitro colony formation assays. ALL-1 double knockout ES cells formed a significantly greater number of colonies with faster kinetics than wild-type and ALL-1 single knockout ES cells. Parental ES cells formed lineage-restricted colonies, whereas single and double knockout ES cells developed, at high frequency, immature and/or “biphenotypic” colonies, mimicking the aberrant hematopoiesis typical of leukemic patients. These data are consistent with the possibility that loss of function of the ALL-1 gene is important in leukemogenesis.

Aberrant FHIT Transcripts in Merkel Cell Carcinoma

Abstract: Merkel cell carcinoma is a rare neuroendocrine carcinoma of the skin which shares several features with small cell lung carcinoma. In a previous study, we reported a high frequency of abnormalities of the FHIT gene, located at 3p14.2, in small cell lung tumors. To determine the role of the FHIT gene in small cell neuroendocrine malignancies, 14 cases of Merkel cell carcinoma were analyzed by reverse transcription of FHIT mRNA followed by PCR amplification and sequencing of products. Eight of 14 tumors (57%) displayed abnormal FHIT products that lacked three or more exons of the FHIT gene. The pattern of abnormal transcripts was similar to that observed in small cell lung tumors, suggesting that FHIT abnormalities might be a common genetic marker of these two types of neuroendocrine tumors.

Complete exon structure of the ALL1 gene

Abstract: The ALL1 gene is found rearranged in approximately 10% of acute lymphoblastic leukemias and in over 5% of acute myeloid leukemias. The gene undergoes fusion with either a variety of partner genes located on different chromosomes or with itself. To further characterize the role of the ALL1 gene in the leukemogenic process, and possibly in solid malignancies, we defined its complete genomic structure. The gene, which spans a region on chromosome band 11q23 approximately 90 kb in length, consists of 36 exons, ranging in size from 65 bp to 4249 bp. The determination of intronic sequences flanking the exon boundaries will allow the determination of whether point mutations may be responsible for inactivation of the gene in solid tumors showing loss of heterozygosity at region 11q23.

Methods for screening and treating leukemias resulting from all-1 region chromosome abnormalities

Abstract: The cDNA sequence of the ALL-1 gene on chromosome 11 is provided. A partial sequence of the AF-4 gene is also provided in the context of the sequences of two reciprocal endproducts of a translocation. Amino acid sequences corresponding to the cDNA sequences of the entire ALL-1 gene and the partial sequence of the AF-4 gene, and sequences relating to chimeric genes formed by chromosome translocations with chromosome 4, 9 and 19, respectively, are provided. Probes are provided for detecting chromosome abnormalities involving the ALL-1 gene on chromosome 11, including probes for detecting chimeric genes generated by translocations. Monoclonal antibodies for diagnosis and treatment and antisense oligonucleotides for treatment of acute leukemias are also described.

Characterization of t(11;14) translocation in mantle cell lymphoma by fluorescent in situ hybridization.

Abstract: Characterization of chromosome abnormalities in leukemia and lymphoma have contributed to the understanding of the molecular basis of these neoplastic diseases. In addition, specific chromosomal aberrations have acquired diagnostic or prognostic value. The t(11;14)(q13;q32) chromosome translocation has been detected in mantle cell lymphomas. However, possibly due to the limits of conventional cytogenetic analysis and the presence of different breakpoints at the molecular level, it is possible that the true percentage of association is underestimated. In our study, we used a yeast artificial chromosome, spanning the entire area where the rearrangements occur on chromosome 11q13, to detect the presence of translocations by fluorescent in situ hybridization experiments. We detected BCL-1 translocations in eight of eight patients with clinical and immunological features of mantle cell lymphoma, suggesting that the t(11;14) translocation is a critical event in the pathogenesis of MCL and may be a primary element for the diagnosis. Since this translocation is associated with poor prognosis, its detection may help to make a correct diagnosis as well as to evaluate residual disease, which is critical to plan a rational chemotherapy regimen.

Modulation of bcl-2 phosphorylation

Abstract: Methods of identifying compounds that modulate bcl-2 mediated cell death are disclosed. The methods comprise contacting a cell or cell extract with a test compound and detecting the level of bcl-2 phosphorylation compared to the level of bcl-2 phosphorylation in a similar cell or cell extract not contacted with the test compound. Methods of treating an individual susceptible to or suffering from a disease characterized by inhibition of apoptosis are disclosed. The methods comprise administering to such an individual an bcl-2 phosphorylation compound that inhibits dephosphorylation of bcl-2 and/or facilitates phosphorylation of bcl-2. Methods of treating an individual susceptible to or suffering from a diseases characterized by apoptosis are disclosed. The methods comprise administering to such an individual a bcl-2 dephosphorylation compound that inhibits phosphorylation of bcl-2 and/or facilitates dephosphorylation of bcl-2.

Fragile histidine triad (FHIT) nucleic acids and methods of producing FHIT proteins

Abstract: The present invention relates to nucleotide sequences of FHIT genes and amino acid sequences of their encoded proteins, as well as derivatives and analogs thereof, and antibodies thereto. The FHIT gene sequence is mutated in diseases involving cell overproliferation, particularly malignancies of the digestive tract. The present invention further relates to compositions of FHIT nucleic acids and a pharmaceutically acceptable carrier.

The t(6;16)(p21;q22) chromosome translocation in the LNCaP prostate carcinoma cell line results in a tpc/hpr fusion gene.

Abstract: Very little is known about the molecular and genetic mechanisms involved in prostate cancer. Previous studies have shown frequent loss of heterozygosity (40%) at chromosomal regions 8p, 10q, and 16q, suggesting the presence of tumor suppressor genes in these regions. The LNCaP cell line, established from a metastatic lesion of human prostatic adenocarcinoma, carries a t(6;16)(p21;q22) translocation. To determine whether this translocation involved genes important in the process of malignant transformation, we cloned and sequenced the t(6;16) breakpoint of this cell line. Sequence analysis showed that the breakpoint is within the haptoglobin gene cluster on chromosome 16, and that, on chromosome 6, the break occurs within a novel gene, tpc, similar to the prokaryotic S10 ribosomal protein gene. The translocation results in the production of a fusion transcript, tpc/hpr.

Molecular diagnosis of lymphoma.

Abstract: The biologic and clinical heterogeneity of lymphomas represents the major obstacle to their diagnosis. Because histologic analysis, which is the initial diagnostic approach, has been demonstrated to be insufficient in the definition of certain types of lymphomas, molecular and immunologic techniques have been increasingly applied to obtain a precise diagnosis and to establish a correct treatment. Fluorescence in situ hybridization, in particular, is a powerful technique with many applications to the study of chromosomal rearrangements. In addition, because of their specificity and sensitivity, molecular techniques provide an important tool in assessing response to treatment, in detecting minimal residual disease, and in understanding the clinical and prognostic significance of the disease.

Characterization of the TCLI Gene and Its Involvement in T-Cell Malignancies

Abstract: Most human leukemias and lymphomas exhibit non-random chromosomal alterations. Translocations are the most common form of alteration in hematopoetic malignancies, leading to oncogenic activation either by the formation of chimeric transforming genes or by deregulation of transcription (Croce, 1987; Rabbitts, 1994). Gene fusion is observed in the majority of the myeloid tumors and in soft tissue sarcomas, and less frequently in B and T cell leukemias and lymphomas: good examples of these alterations are the t(9;22) translocation of chronic myelogenous leukemia (CML) and Ph chromosome positive acute lymphocytic leukemia (Ph+ALL) and the t(1;19), t(15;17), t(6;9) and t(4;11) chromosomal translocations observed in pre-B, myeloid and mixed lineage leukemias, all of which generate chimeric transcripts (Nourse et al., 1990, Kamps et al., 1990, and Gu et al. 1992). Deregulation of oncogene transcription is observed in most B and T cell leukemias and lymphomas, in these alterations the immunonoglobulin (Ig) or the T-cell-receptor (TCR) loci become juxtaposed to cellular protoncogenes such as c-MYC, BCL-2, TCL-2, BCL-1, TCL-3 (for a review see Croce, 1987; Rabbitts, 1994). In this latter case the gene justaxposed to the Ig or the TCR locus can be quite close (within 10–30 kb) from the genomic breakpoint, as for BCL-2 on chromosome 18q21, BCL-3 on chromosome 19q13.1, c-MYC on 8q24 in sporadic cases of Burkitt Lymphomas (BLs),or far (up to few hundreds kilobases) as for BCL-1 on chromosome 11q13, and c-MYC, in the endemic cases of BLs. These translocations are belived to be due to errors during the physiological process of the Ig and TCR gene rearrangements, and result in the activation of oncogenes by their juxtaposition to regulatory elements (enhancers) of the Ig or TCR loci.