Identification and sequencing of the Syrian Golden hamster (Mesocricetus auratus) p16(INK4a) and p15(INK4b) cDNAs and their homozygous gene deletion in cheek pouch and pancreatic tumor cells.

Previous studies have shown that the p16(INK4a) tumor suppressor gene is inactivated in up to 98% of human pancreatic cancer specimens and 83% of oral squamous cell carcinomas. Inactivation of the related p15(INK4b) gene has also been identified in a number of tumors and cell lines, however, its role as an independent tumor suppressor remains to be elucidated. Chemically-induced tumors in the Syrian Golden hamster (Mesocricetus auratus) have been shown to be excellent representative models for the comparative development and progression of a number of human malignancies. The purpose of this study was to determine the importance of the p16(INK4a) and p15(INK4b) genes in two experimental hamster models for human pancreatic and oral carcinogenesis. First, hamster p16(INK4a) and p15(INK4b) cDNAs were cloned and sequenced. The hamster p16(INK4a) cDNA open reading frame (ORF) shares 78%, 80%, and 81% identity with the human, mouse, and rat p16(INK4a) sequences, respectively. Similarly, the hamster p15(INK4b) cDNA ORF shares 82% and 89% sequence identity with human and mouse p15(INK4b), respectively. Second, a deletion analysis of hamster p16(INK4a) and p15(INK4b) genes was performed for several tumorigenic and non-tumorigenic hamster cell lines and revealed that both p16(INK4a) and p15(INK4b) were homozygously deleted in a cheek pouch carcinoma cell line (HCPC) and two pancreatic adenocarcinoma cell lines (KL5B, H2T), but not in tissue matched, non-tumorigenic cheek pouch (POT2) or pancreatic (KL5N) cell lines. These data strongly suggest that homozygous deletion of the p16(INK4a) and p15(INK4b) genes plays a prominent role in hamster pancreatic and oral tumorigenesis, as has been well established in correlative studies in comparable human tumors. Furthermore, this study supports the comparative importance of the hamster pancreatic and cheek pouch models of carcinogenesis in subsequent mechanistic-, therapeutic-, and preventive-based studies aimed at providing important translational data applicable to pancreatic adenocarcinoma and oral squamous cell carcinoma in humans.

Responsiveness to transforming growth factor-beta (TGF-beta)-mediated growth inhibition is a function of membrane-bound TGF-beta type II receptor in human breast cancer cells.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-beta are mediated by the TGF-beta receptor complex, a multimer composed of TGF-beta receptor type I (TbetaR-I) and TGF-beta receptor type II (TbetaR-II) subunits. Evidence suggests that loss of expression of Tbeta3R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-beta-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-beta-induced G1 growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-beta1 and a significant binding of 125I-labeled TGF-beta ligand. While expression of TbetaR-I mRNA was similar across the panel of cell lines, TbetaR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF- 10F cell line. When total cellular protein was fractionated by centrifugation, TbetaR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbetaR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-beta-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbetaR-II protein appears to be an important determinant of resistance to TGF-beta-mediated growth inhibition in this group of breast cell lines.

Analysis of TGF-beta type I receptor for mutations and polymorphisms in head and neck cancers.

Transforming growth factor-beta receptor (TbetaR)-dependent signals are critical for cell growth and differentiation and are often disrupted during tumorigenesis. The entire coding region of TbetaR-I and flanking intron sequences from 30 head and neck carcinomas were examined for alterations using "Cold" SSCP and direct sequencing. No somatic point mutations were found in the TbetaR-I gene. In contrast, 14 polymorphic sequence changes were detected in TbetaR-I in 13 (43%) of the samples, including eight (27%) nucleotide alterations identified as polymorphisms in an exon-1 (GCG)(9) microsatellite repeat, a previously reported tumor susceptibility allele. A nine base pair deletion was found in 23% of the samples including five heterozygous and two homozygous deletions as well as single homozygous 12bp deletion. Additionally, six heterozygous polymorphisms in intronic sequences were determined, including one heterozygous C/A genotype at the +82 nucleotide position of the intron-5 intervening sequence (IVS), and five heterozygous G/A genotypes within intron-7 at the +24 nucleotide position. Exon-1 polymorphisms in the (GCG)(9) microsatellite region of the TbetaR-I gene and their association with head/neck cancers, suggest that development of these cancers may be a direct consequence of loss of responsiveness to TGF-beta mediated growth inhibition.

Frequent mutation of p16 in squamous cell carcinoma of the head and neck.

RNA was isolated from 22 squamous cell carcinomas (SCCs) obtained from diverse sites within the head and neck and from matched normal tissue where available. Tissue samples were then screened for expression of RNA from tumor suppressor gene p16 by utilizing semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis. p16-Specific PCR amplification products generated from tumor samples were subject to further analysis by direct DNA sequencing to determine if any tumor sample harbored a p16 mutation. The results show the presence of mutations in 10 of 22 (45%) of the tumor samples. Mutations comprise two identical point mutations, two small deletions (1 bp and 2 bp), one single-nucleotide insertion, four larger deletions, and an insertion/deletion. No mutations in p16 have been identified by analysis of PCR products generated from normal matched tissue, suggesting that p16 alterations are generated by somatic mutation and are not germline in origin. All 22 samples were analyzed additionally by immunohistochemistry for nuclear expression of the retinoblastoma (RB) tumor suppressor gene product. Results show lack of RB nuclear expression in only one sample, suggesting that mutation of RB is an infrequent event in the development of SCC of the head and neck (SCCHN).

GRS, a novel member of the Bcl-2 gene family, is highly expressed in multiple cancer cell lines and in normal leukocytes.

Our laboratory previously described the independent isolation of the fibroblast growth factor 4 (FGF-4) gene by NIH3T3 transformation assay using DNA from a patient with CML leukemia (Lucas et al., 1994). The FGF-4 gene was truncated by DNA rearrangement with a novel gene named GRS. In this manuscript we describe isolation of GRS cDNA and show by sequence comparison that GRS is a novel member of the Bcl-2 gene family. Northern analysis shows expression of the gene in normal human tissue to be largely restricted to the hematopoietic compartment. Analysis of the pattern of gene expression in cancer cell lines demonstrates GRS is expressed in hematopoietic malignancies and in melanoma. The chromosomal location of GRS has also been determined. The gene is positioned on chromosome 15 within bands q24-25.

Multiple phosphotyrosine phosphatase mRNAs are expressed in the human lung fibroblast cell line WI-38.

Protein tyrosine phosphatases are important components of signal transduction pathways. The authors have used reverse transcription/polymerase chain reactions to accomplish a comprehensive examination of the RNA expression for 58 distinct mammalian protein tyrosine and dual specificity phosphatase (PTPase) and PTPase-like genes in the normal human diploid fibroblast cell line WI-38. Thirty-seven of these PTPase genes express easily measurable RNA, and four simultaneously express the RNA for two or more isoforms. Messages for an additional eight PTPase genes are detectable at low levels. Only 14 known PTPase genes do not express measurable RNA under our conditions. For purposes of comparison, the authors also assessed the PTPases expressed in the WI-38 cell line using highly degenerate primers to conserved motifs found in the classical tyrosine-specific PTPases. Only eight of the 22 classic tyrosine-specific PTPases detected using the specific primers were detected using these degenerate primers. Our panel of specific PTPase primers should be very useful for semiquantitatively assessing the repertoire of PTPases expressed by cells.

Regulation of FGF-4 enhancer activity by transcription factor NF-Y.

Regulation of FGF-4 gene expression is controlled both by elements in the promoter and by an enhancer domain located in the untranslated region of the third exon. We have determined that transcription factor NF-Y binds to the FGF-4 promoter. We further show by mutational analysis that binding of NF-Y is essential for FGF-4 enhancer activity but has minimal effect on activity of the FGF-4 promoter alone.

Differential expression of cytokine genes in HIV-1 tat transfected T and B cell lines.

Cytokine responses are dramatically affected when HIV-1 infected cells are activated with certain antigenic stimuli. We report the effects of HIV-1 tat gene in cytokine modulation, using HIV-1 tat transfected T (Jurkat) and B (Raji) cell lines. Studying the effect of tat and/or PMA + PHA on mRNA expression of 14 cytokines (IL-1 alpha, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNF-alpha, TNF-beta, GM-CSF, TGF-beta, IFN-gamma and MIP-1 alpha) illustrated differential effects. In addition to the varied effects of tat on the steady state levels of cytokine mRNAs, tat induced the secretion of TNF-beta preferentially in both B and T cell lines, either by itself as in Raji B cell line or synergistically upon PMA + PHA stimulation as in Jurkat T cell line.

B cell IL-7. Human B cell lines constitutively secrete IL-7 and express IL-7 receptors.

IL-7 was originally reported as a cytokine produced by stromal cells which supports pre-B cell proliferation in vitro. To determine whether human B cells secrete IL-7 and express IL-7R we studied a wide panel of B cell lines (CLS). In Northern blot analysis we detected 2.4 kb IL-7 mRNA and by quantitative PCR we demonstrated IL-7 expression in 5 of 6 B CLS derived from patients with AIDS-associated Burkitt's lymphoma (AABCL), 3 of 3 CLS derived from patients with American Burkitt's lymphoma and 5 of 6 normal lymphoblastoid CLS. Only 1 of 5 African Burkitt's lymphoma CLS and 2 of 7 EBV- CLS expressed IL-7. A total of 484-bp amplicons was cloned and sequenced and found to correspond to the original IL-7 sequence. Constitutive IL-7 secretion was detected in 5 of 6 AABCL and in 6 of 7 normal lymphoblastoid CLS but in none of the 7 EBV- CLS. IL-7R expression was demonstrated in 8 of 26 CLS, none of which secreted IL-7. Our data suggest that 1) IL-7 mRNA is expressed in malignant B cell phenotypes, which correspond to a narrow window in the B cell differentiation pathway (pre-B, early B, intermediate B), as well as in normal lymphoblastoid B CLS. 2) IL-7 mRNA is expressed in both EBV+ and EBV- CLS, but only the EBV+ CLS secrete IL-7. 3) B cells activated by both EBV and HIV-1 (AABCL) secrete the greatest amount of IL-7. 4) IL-7 autocrine loops are not evident since IL-7R were detected on on CLS, which do not secrete IL-7. Our data provide the first direct evidence of IL-7 secretion by human cells and it is yet to be determined whether IL-7 is secreted by other cell types.

Human B cell lines express the interferon gamma gene.

Interferon gamma (IFN-gamma) production has been attributed exclusively to activated T cells and NK cells. We sought to determine whether human B cells express IFN-gamma. We studied 28 B cell lines including Epstein-Barr virus (EBV)+ normal lymphoblastoid B cell lines (N = 7), EBV+ B cell lines derived from patients with Burkitt's lymphoma with (N = 6) or without AIDS (N = 8), as well as seven EBV- B cell lines. All cell lines were studied by reverse transcription-polymerase chain reaction (RT-PCR). We detected constitutive expression of IFN-gamma in every B cell line. The tumor promoters PMA and teleocidin appeared to enhance this IFN-gamma expression in nearly every B cell line. The 517 bp amplicons spanning the entire protein coding region of the IFN-gamma mRNA from three representative lines were sequenced, definitively establishing that B cell IFN-gamma is identical to IFN-gamma from activated T cells and is not altered by derivation of the B cell lines from AIDS patients or by EBV status. Detection of IFN-gamma in the entire panel of EBV+ and EBV- cell lines suggests that the IFN-gamma gene is broadly expressed by human B cells. Our data imply that human B cells can be activated to produce IFN-gamma, further enmeshing B cells in the dynamics of immunoregulation.

Human B-cell interleukin-10: B-cell lines derived from patients with acquired immunodeficiency syndrome and Burkitt's lymphoma constitutively secrete large quantities of interleukin-10.

A recent addition to the lymphokine network is human IL-10 (hIL-10). This novel lymphokine has striking homology to BCRF1 protein, the product of a previously uncharacterized open-reading frame in the Epstein-Barr virus (EBV) genome. To date, IL-10 expression has been described in several T clones induced with anti-CD3 and phorbol myristate acetate (PMA), in monocytes stimulated with lipopolysaccharide (LPS), and in murine B-cell lymphomas. We sought to determine whether human B cells express hIL-10 and, if so, its relationship to EBV and to other B-cell lymphokines. We studied 21 EBV-positive B-cell lines derived from patients with acquired immunodeficiency syndrome (AIDS) and Burkitt's lymphoma (n = 6), American Burkitt's (n = 3), African Burkitt's (n = 5), and normal lymphoblastoid cell lines (n = 7), in comparison with seven EBV-negative cell lines. All cell lines were activated with the tumor promoters PMA and teleocidin and were studied by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoadsorbent assay (ELISA). We demonstrated that EBV-positive cell lines derived from patients with American Burkitt's lymphoma, and especially those from patients with AIDS, constitutively express large quantities of hIL-10 by Northern blot analysis and ELISA (range, 3,101 to 25,915 pg/mL), and that both teleocidin and PMA induce hIL-10 in these cell lines. In contrast, six of seven EBV-negative cell lines did not express hIL-10 even by RT-PCR, and hIL-10 was not triggered by PMA or teleocidin. To assure that the 350 bp amplified by PCR was hIL-10 and not BCRF1, we used PCR primers, which do not amplify a fragment from plasmid templates containing BCRF1. Cloning and sequencing of the 350 bp product also demonstrated that B-cell IL-10 is identical to hIL-10 from the T-cell clone B21. Correlation of hIL-10 with other B-cell lymphokines secreted by these B-cell lines demonstrated that hIL-10 secretor cell lines also constitutively secrete or can be induced to secrete IL-6, although to a much lesser amount. Since both lymphokines influence B-cell growth and differentiation, we suggest that hIL-10 may contribute to the polyclonal B-cell activation and hyperglobulinemia seen in AIDS patients. Finally, several reports support the hypothesis that EBV is an important cofactor in the development of human immunodeficiency virus type 1 (HIV-1)-related B-cell lymphomas. Detection of large quantities of hIL-10 in B-cell lines derived from AIDS patients, the close association between EBV and hIL-10 shown in this report, and the ability of BCRF1 to capture hIL-10 activities, make hIL-10/BCRF1 an attractive candidate as a factor causing B-cell growth and immortalization in patients with AIDS and B-cell lymphomas.