Sherry A. Salzman

Analysis of Candidate Genes for Prostate Cancer

Considerable evidence demonstrates that genetic factors are important in the development and aggressiveness of prostate cancer. To identify genetic variants that predispose to prostate cancer we tested candidate SNPs from genomic regions that show linkage to prostate cancer susceptibility and/or aggressiveness, as well as genes that show a significant difference in mRNA expression level between tumor and normal tissue. Cases had histologically verified prostate cancer. Controls were at least 65 years old, never registered a PSA above 2.5 ng/ml, always had digital rectal examinations that were not suspicious for cancer, and have no known family history of prostate cancer. Thirty-nine coding SNPs and nine non-coding SNPs were tested in up to 590 cases and 556 controls resulting in over 40,000 SNP genotypes. Significant differences in allele frequencies between cases and controls were observed for ID3 (inhibitor of DNA binding), p = 0.05, HPN (hepsin), p = 0.009, BCAS1 (breast carcinoma amplified sequence 1), p = 0.007, CAV2 (caveolin 2), p = 0.007, EMP3 (epithelial membrane protein 3), p < 0.0001, and MLH1 (mutL homolog 1), p < 0.0001. SNPs in three of these genes (BCAS1, EMP3 and MLH1) remained significant in an age-matched subsample.

PCR analysis of nasal polyps, chronic sinusitis, and hypertrophied turbinates for DNA encoding bacterial 16S rRNA.

Background Nasal polyps are considered to result from chronic inflammation, but the initial or persisting stimulus for the inflammation is not known. A variety of bacteria and fungi have been cultured from nasal polyps, but ∼35% have sterile cultures. Previously, Mycoplasma pneumoniae–specific DNA was detected in human nasal polyps using polymerase chain reaction (PCR) techniques, suggesting M. pneumoniae as a causative agent in the etiology of nasal polyps. Methods In this study, we tested for the presence of bacterial DNA in nasal polyps resected from 40 patients, in nasal mucosa membrane from 9 patients undergoing turbinectomy for hypertrophy, and in sinus mucosa membrane from 6 patients undergoing endoscopic surgery for chronic sinusitis. Tissue DNA was extracted and analyzed by PCR using M. pneumoniae specific primers for DNA that encode the 16S rRNA gene in 41 specimens (31 polyps, 6 turbinates, and 4 sinus), and by consensus sequence-based PCR using broad range primers for most eubacterial DNA encoding the 16S rRNA gene in 38 specimens (26 polyps, 7 turbinates, and 5 sinuses). Results Only two samples were positive for bacterial DNA encoding 16S rRNA: Streptococcus sp. DNA was isolated from one polyp specimen and Pseudomonas aeruginosa DNA was isolated in one maxillary sinusitis specimen. No evidence of M. pneumoniae–specific DNA encoding 16S rRNA was found in any of the tissues. Conclusions This study suggests that chronic bacterial infection is not a major component of nasal polyp etiology.

Transforming growth factor-β and multidrug resistance in chronic lymphocytic leukemia

Patients with chronic lymphocytic leukemia (CLL) frequently respond to initial treatment, but then become resistant to chemotherapy. Studies have shown one important cause of chemotherapeutic resistance to be multidrug resistance (MDR). To investigate the potential role of MDR and transforming growth factor-β (TFG-β), a potent growth inhibitor of B lymphocytes, in the development of chemotherapeutic resistance in CLL, we evaluated 22 CLL patients for loss or mutation of TGF-β receptors (TβR), plasma TGF-β1 levels, and expression of MDR1 mRNA. Receptor crosslinking and immunoprecipitation experiments did not demonstrate loss of TβRs in any patients studied. No relationship between plasma TGF-β1 levels and expression of MDR1 mRNA was seen. Correlation of plasma TGF-β1 levels to disease stage revealed a consistent decline in plasma TGF-β1 levels with advancing disease stage (P=0.031).

Regulation of colony-stimulating factor-induced human myelopoiesis by transforming growth factor-β isoforms

Transforming growth factor-beta (TGF-beta) proteins are multifunctional regulators of cell growth and differentiation. The three isoforms, TGF-beta1, -beta2, -beta3 share approximately 70% identical amino acid sequence and are coded by three distinct genes. Growth and differentiation functions in which the isoforms have differential activity include: inhibition of colorectal cancer cell growth, migration of aortic endothelial cells, survival of ciliary ganglionic neurons, and binding to cell surface receptors. A previous paper reported that TGF-beta1 and TGF-beta2 had bimodal dose-dependent stimulatory and inhibitory effects on granulocyte-macrophage colony-stimulating factor induced Day 7 granulocyte-macrophage colony-forming units. The effects of TGF-beta3 were only inhibitory. At low concentrations, TGF-beta1 and -beta2 stimulated growth, whereas at higher concentrations both isoforms inhibited growth. We now report that TGF-beta1, TGF-beta2, and TGF-beta3 are similar to each other at low concentrations; at higher concentrations TGF-beta1 and TGF-beta3 inhibit growth, but TGF-beta2 stimulates growth. Our results are consistent with the known affinities of the TGF-beta isoforms with the Type II TGF-beta signaling receptor, which has greater affinity for TGF-beta1 and TGF-beta3 than TGF-beta2.

Identification of a gene frequently mutated in prostate tumors

Although prostate cancer is the second leading cause of cancer death for men in the United States, the genetics of tumor development are poorly understood. Several expressed sequence tagged genes (ESTs) that are expressed predominantly in the prostate have recently been identified, although their role in the development and maintenance of the prostate is unknown. Here, we demonstrate that the gene identified as UNIGENE cluster Hs. 104215, which codes for a message found predominantly in the prostate, may be important in tumor development. We name this gene PCan1 for Prostate Cancer gene 1. Northern blot experiments were performed using RNA isolated from tumor-derived cell lines and human prostate to determine the expression pattern of the gene. DNA sequencing was used to identify mutations that occurred in tumor tissue. By Northern blot analysis, this gene product was not detectable in LNCaP, DU 145, or PC-3 prostate cancer cell lines, although it was readily observed in RNA isolated from total prostate and from dissected central and peripheral regions of prostate. Sequence analysis of genomic DNA from LNCaP, DU 145, or PC-3 cells demonstrated a G/A polymorphism at position 193. Analysis of matched tumor-derived DNA and blood-derived DNA samples from 11 of 13 patients who had undergone a radical prostatectomy and who were homozygous for A in blood-derived DNA demonstrated mutation of position 193 in matched tumor samples resulting in G/A polymorphism. Sixteen additional patient samples were G/A polymorphic in both blood-derived DNA and tumor-derived DNA and two samples were GG in both blood-derived and tumor-derived DNA. Our results suggest that this gene may be a hot spot for mutation in prostate cancer, especially because our radiation hybrid mapping located this gene within a region identified in linkage mapping studies of affected families with prostate cancer. Loss of heterozygosity in prostate tumors has also been reported at the location of PCan1. Further studies to determine the functional role of this candidate tumor suppressor gene are warranted.

Expression and initial promoter characterization of PCAN1 in retinal tissue and prostate cell lines

Prostate cancer is the most frequently diagnosed neoplasia in men and one of the leading causes of cancer-related deaths in men over 60. In an effort to understand the molecular events leading to prostate cancer, we have identified PCAN1 (prostate cancer gene 1) (also known as GDEP), a gene that is highly expressed in prostate epithelial tissue and frequently mutated in prostate tumors. Here we demonstrate its expression in neural retina, and retinoblastoma cell culture but not retinal pigment epithelial cell culture. We further characterize PCAN1 expression in the prostate cell lines RWPE1, RWPE2, and LnCAP PGC. We demonstrate an increase in expression when the cells are grown in the presence of Matrigel, an artificial extracellular basement membrane. Expression was time dependent, with expression observed on d 6 and little or no expression on d 12. Testosterone was not found to increase PCAN1 expression in this culture system. In addition, normal prostate epithelial cells co-cultured with normal prostate stromal cells did not exhibit PCAN1 expression at any time. To definitively locate the transcription initiation sites, we performed restriction-ligase-mediated 5′ RACE, to selectively amplify only mRNA with a 5′ cap. An initial characterization of the sequence upstream of the initiation sites determined six possible binding sites for the prostate specific regulatory protein NKX3. 1 and four potential binding sites for the PPAR/RXR heterodimer that is involved in the control of cell differentiation and apoptosis.

Amino acids 67 and 68 of transforming growth factor-β regulate binding to a glycosyl phosphatidyl inositol-linked membrane protein on vascular endothelial cells

Transforming growth factor-beta (TGF-beta) is a multifunctional growth and differentiation factor that affects almost all cells. Although equipotent in many cases, the three isoforms of TGF-beta (-beta1, -beta2, -beta3) have several important isoform specific activities. For example, TGF-beta2 binds with higher affinity to a 60 kDa cell-surface glycosyl phosphatidylinositol (GPI)-linked protein, expressed on vascular endothelial cells. We used chimeric TGF-beta proteins, in which selected regions of TGF-beta1 had been exchanged for the corresponding region of TGF-beta2, to demonstrate that amino acids 67 and 68 regulate binding of TGF-beta to this protein. Exchange of amino acids 67 and 68 of TGF-beta1 into TGF-beta2 resulted in a protein similar in affinity to TGF-beta1 for binding to the GPI-linked protein. In contrast, exchange of only amino acid 67 of TGF-beta1 into TGF-beta2, or exchange of only amino acid 68 of TGF-beta1 into TGF-beta2, resulted in a protein with affinity similar to that of TGF-beta2. This suggests that the coordinated change of Gln and His of TGF-beta1 to Thr and Ile at positions 67 and 68 alters the specificity of TGF-beta. Amino acids 67 and 68 are part of a surface-exposed alpha-helix that forms a projection away from the center of the TGF-beta molecule and is accessible for receptor binding.

Small molecule antagonists of the TGF-β1/TGF-β receptor binding interaction

Excessive and inappropriate action of transforming growth factor (TGF)-β has been implicated in the pathogenesis of several disease processes, especially cancer and fibrosis. To identify antagonists of the TGF-β ligand-binding domain that may have therapeutic potential, we screened the National Cancer Institute open access chemical repository for molecules that inhibited binding of TGF-β to the type II receptor (TβRII). About 30,000 molecules were screened resulting in the identification of five structurally related molecules that reduced binding of TGF-β1 to soluble TβRII with an ED50 of approx 10 μM. The chemicals blocked inhibition of Mv1Lu cell growth by TGF-β, TGF-β-induced expression of luciferase driven by the TGF-β response element, and induction of plasminogen inhibitor mRNA detected by Northern blot. In contrast, the chemicals did not block activin-induced inhibition of cell growth. Our results identify a novel chemical group that blocks binding of TGF-β to its receptor and may result in novel treatment for disease.