Linear amplification of gene-specific cDNA ends to isolate full-length of a cDNA.
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Blotting, Southern↗
Biomedical subjects
M J Becich
Publications and source records attributed to M J Becich.
Expression patterns of vitamin D receptor in human prostate.
UV exposure and serum levels of vitamin D have been linked in several studies with prostate cancer risk. At the cellular level, the principal action of vitamin D is mediated though vitamin D receptors (VDR). Since prostate cancer is a disease strongly associated with age, we examined the presence of VDR in normal prostate from donors of various ages to determine if the VDR expression pattern changed with age. We also compared the VDR expression in the peripheral and central zones of the prostate to determine if the expression pattern varied by location. Immunohistochemical studies were performed on paraffin-embedded tissue from cases selected by the following age decades; 10-19, 20-29, 30-39, 40-49, 50-59, and 60-69. Both the central and peripheral zones were examined for VDR expression. The intensity of VDR expression in prostate was compared with expression in different types of human tissues. Mean VDR expression was lowest in the 10-19 years of age group. The intensity of the nuclear VDR was higher though the fifth decade, and then declined in cases of ages 60-70. When multiple sections of the same donor prostate were compared, VDR expression was greater in the peripheral zone compared to the central zone.
Adolescent↗
E-cadherin expression and PSA secretion in human prostate epithelial cells.
Prostate-specific antigen (PSA) is the most widely used marker for the diagnosis of prostate cancer and is an independent predictor of prostatic capsular invasion. A number of studies have identified E-cadherin, a cell adhesion protein, as a potential invasion suppressor which is decreased in prostate adenocarcinoma. Our goal in the present study was to evaluate E-cadherin expression in primary cultures and determine the relationship between E-cadherin expression and PSA secretion in both primary cultures and the prostate tumor cell line, LNCaP. Immunohistochemical studies and Western blot analysis confirmed greater expression of E-cadherin in normal epithelial cells than tumor-derived prostate cells. This is the first report that the incubation of normal prostate epithelial cells with E-cadherin antibody increases the amount of PSA detected in the media of normal cells as well as in LNCaP. Since E-cadherin may function as an invasion suppressor, an understanding of the decreased expression of this adhesion factor and the impact on PSA secretion may aid in understanding epithelial tumorigenesis.
Antibodies↗
Myopodin, a synaptopodin homologue, is frequently deleted in invasive prostate cancers.
Prostate cancer is one of the leading causes of cancer-related deaths for men in the United States. Like other malignancies, prostate cancer is underscored by a variety of aberrant genetic alterations during its development. Although loss of heterozygosity or allelic loss is frequently identified among prostate cancers, few genes have been identified thus far as critical to the development of invasive prostate cancers. In this report, we used the recently developed technology, the "differential subtraction chain," to perform a genome-wide search for sequences that are deleted in an aggressive prostate cancer. Among the deleted sequences, we found that one sequence was deleted in >50% of prostate cancers we tested. We mapped this sequence to chromosome 4q25 by screening the Genebridge 4 hamster radiation panel with primers specific to this probe, and subsequently identify a 54-kb minimal common deletion region that contains the sequence encoding myopodin. Sequence analysis indicates that myopodin shares significant homology with synaptopodin, a protein closely associated with podocyte and neuron differentiation. Further study shows that frequent complete or partial deletions of the myopodin gene occurred among invasive prostate cancer cases (25 of 31 cases, or 80%). Statistical analysis indicates that deletion of myopodin is highly correlated with the invasiveness of prostate cancers, and thus may hold promise as an important prognostic marker for prostate cancers.
Amino Acid Sequence↗
The role of the pathologist as tissue refiner and data miner: the impact of functional genomics on the modern pathology laboratory and the critical roles of pathology informatics and bioinformatics.
This article provides an overview of how functional genomics is likely to impact on the pathology laboratory and highlights how informatics and tissue banking will greatly facilitate the molecular age of medicine. Important aspects of functional genomics in the post-genome era, including the roles of laser capture microdissection, DNA- and complementary DNA-based microarrays, proteomic methods, collaborative human tissue banking, tissue microarrays, and pathobioinformatics in the modern pathology laboratory are discussed. The role of mass spectroscopy in the analysis of RNA, DNA, and protein and its impact on the clinical laboratory, particularly in cost-effectiveness and time savings, are evaluated. This article explores how laboratory information systems (LISs) and the devices that feed them information may need to be modified to adapt to greater volumes of data for the new testing modalities that require understanding sophisticated fluorescence detection methods and image processing. Emerging genomic testing methods and their impact on pathology laboratory testing, especially in the area of molecular classification of neoplasms, are examined. The role of the tissue bank in the modern pathology laboratory as an archive of control normal tissues, as well as subsamples of the spectrum of progressive neoplastic states, is discussed in light of its critical importance to the molecular classification of cancer. Establishing a database that combines structured reports in pathology LISs and construction of tissue banking information systems will provide a rich resource for pathology departments. The article discusses a hypothetical resource, such as the Shared Tumor Expression Profiler, that would provide access to well-characterized tissue-based research resources for clinicians and researchers. Last, the article emphasizes how LISs can prepare for these changes, and how training pathologists in pathology informatics and bioinformatics (pathobioinformatics) is critical to ensure pathology's overall leadership role in the post-genome era.
Clinical Laboratory Techniques↗
Expression of cadherins and catenins in paired tumor and non-neoplastic primary prostate cultures and corresponding prostatectomy specimens.
Cadherins are a family of transmembrane proteins that play a crucial role in cell differentiation, cell migration, and intercellular adhesion. Cadherins are associated with catenins through their highly conserved cytoplasmic domain. Down-regulation of E-cadherin protein has been shown in various human cancers. This study examined the expression of cadherins and associated catenins at the mRNA level. Paired tumor and nonneoplastic primary prostate cultures were obtained from surgical specimens. Quantitative multiplex fluorescence reverse transcriptase-polymerase chain reaction (QMF RT-PCR) and quantitative analysis were performed and correlated with immunostain results. Six of seven cases of neoplastic cultures showed moderately-to-markedly decreased levels of E-cadherin and P-cadherin mRNA. Similar losses of alpha-catenin and beta-catenin mRNA were also observed. The results of QMF RT-PCR showed good correlation with the results of immunohistochemical studies based on corresponding formalin-fixed sections. In conclusion, this paper presents a coordinated down-regulation in the expression of E-cadherin and associated catenins at the mRNA and protein level in most of the cases studied. This down-regulation may play an important role in the pathogenesis of prostate cancer.
Adenocarcinoma↗
Detection of bladder cancer using a novel nuclear matrix protein, BLCA-4.
We have identified previously six nuclear matrix proteins (NMPs) that are bladder cancer specific. In this study, we analyzed the expression of one of these proteins, BLCA-4, in bladder tumors and normal bladder tissue. We also examined the appearance of BLCA-4 in the urine as a biomarker for bladder cancer. BLCA-4 was isolated from nuclear matrix preparations of bladder tumors, and its peptide sequence was determined. The antibodies generated against the resulting BLCA-4 peptides were then used to detect its presence in immunoblots and in urine samples by immunoassay. We analyzed tissue samples of bladder tumor and normal donor bladders and urine obtained from 51 normal individuals and 54 patients with pathologically confirmed bladder cancer. The BLCA-4 peptide sequences do not resemble any known human protein sequences. On immunoblot analysis, BLCA-4 expression was detectable in tumor and normal tissues from patients with bladder cancer but not in any of the normal bladder tissue obtained from organ donors. Using a prospectively determined cutoff level of 13 A (absorbance) units/microg protein, all 51 normal individuals tested were negative for BLCA-4 expression, whereas 53 of 55 samples from patients with bladder cancer were positive. These results suggest that BLCA-4 is present throughout the bladder in both the tumor and morphologically normal areas in bladder cancer patients. BLCA-4 is a very sensitive (96.4%) and specific (100%) marker for bladder cancer. BLCA-4 is a bladder cancer-specific marker that can be detected using a urine-based assay and can be used in the diagnosis of bladder cancer.
Adult↗
Prototype Web-based continuing medical education using FlashPix images.
Continuing Medical Education (CME) is a requirement among practicing physicians to promote continuous enhancement of clinical knowledge to reflect new developments in medical care. Previous research has harnessed the Web to disseminate complete pathology CME case studies including history, images, diagnoses, and discussions to the medical community. Users submit real-time diagnoses and receive instantaneous feedback, eliminating the need for hard copies of case material and case evaluation forms. This project extends the Web-based CME paradigm with the incorporation of multi-resolution FlashPix images and an intuitive, interactive user interface. The FlashPix file format combines a high-resolution version of an image with a hierarchy of several lower resolution copies, providing real-time magnification via a single image file. The Web interface was designed specifically to simulate microscopic analysis, using the latest Javascript, Java and Common Gateway Interface tools. As the project progresses to the evaluation stage, it is hoped that this active learning format will provide a practical and efficacious environment for continuing medical education with additional application potential in classroom demonstrations, proficiency testing, and telepathology. Using Microsoft Internet Explorer 4.0 and above, the working prototype Web-based CME environment is accessible at http://telepathology.upmc.edu/WebInterface/NewInterface/welcome.html.
Cell Biology↗
Differential subtraction chain, a method for identifying differences in genomic DNA and mRNA.
Identifying the genetic differences between two organisms or cell types has been a major goal in modern biomedical research. Recently, we developed a novel methodology that can rapidly identify the differences between two populations of DNA. This method, termed 'differential subtraction chain' (DSC), is based on a novel 'negative amplification' strategy that converts (amplifiable) tester sequences to counterpart (unamplifiable) driver sequences. The result is a double exponential elimination of amplifiable sequences in the testers, while preserving the sequences in the testers that have no counterpart in the drivers. We applied this methodology to the genome of a glioblastoma cell line. A homozygous deletion was rapidly identified. We extended this technique to identifying the unique sequences in mRNA. Two CDC25 transgene fragments were quickly identified in a cdc25B transgenic mouse. We also applied this methodology to systems with profound differences in mRNA expression. In a 'prostate epithelia subtracting blood cells' DSC reaction, a sample of unique gene fragments which are absent in the prostate but present in the blood were identified. Lastly, we detected rare (1 virus/100 cells) Herpes simplex virus type 2 (HSV-2) sequences in a tissue culture, indicating good sensitivity of this methodology. Overall, DSC represents a fast, efficient and sensitive method for identifying differences in genomic DNA and mRNA and can be easily applied in a variety of biological systems.
Animals↗
Methylation of the CD44 metastasis suppressor gene in human prostate cancer.
Previous studies demonstrated that CD44 is a metastasis suppressor gene for prostate cancer and that the expression of CD44 both at mRNA and protein levels is down-regulated during prostate cancer progression, with down-regulation being correlated with higher tumor grade, aneuploidy, and distant metastasis. In this study, we evaluated DNA hypermethylation as a potential mechanism accompanying this decreased CD44 expression in human prostate cancer. Nucleotide sequence analysis revealed a CpG island in the CD44 transcriptional regulatory region. We found that cytosine methylation of CD44 promoter occurs in CD44-negative prostate cancer cell line (i.e., LNCaP) but not in prostate cancer cell lines (i.e., TSU, PC3, and DU145) expressing this gene. In addition, we examined methylation status of CD44 in 84 matched normal and cancer prostate specimens. Hypermethylation of the 5' CpG island of CD44 gene was observed in 31 of 40 primary prostate cancer specimens, 3 of 4 distant organ site metastases obtained at autopsy from men who died of prostate cancer, and 4 of the 40 matched normal tissues. These results demonstrated that methylation of the 5' CpG island of CD44 gene is closely associated with transcriptional inactivation, resulting in a decreased expression of CD44 in human prostate cancer.
Adenocarcinoma↗
Differential effects of vitamin D on normal human prostate epithelial and stromal cells in primary culture.
OBJECTIVES: Because epidemiologic evidence has demonstrated that vitamin D may play a role in the etiology of prostate cancer, we tested the inhibitory effect of the biologically active form of vitamin D (1,25-D) on the cell proliferation of human prostate epithelial and stromal cells in a chemically defined situation in the presence and absence of dihydrotestosterone (DHT). We also tested the effect of 1,25-D in castrated rats in the presence and absence of flutamide, an androgen receptor blocker. METHODS: Prostate stromal and epithelial cells were isolated from freshly collected human prostatectomy specimens, and cell proliferation was measured with the MTT assay. Immunohistochemistry was performed to detect the presence of 1,25-D receptors, androgen receptors, smooth muscle actin, and E-cadherin. For in vivo analysis of 1,25-D, male Sprague-Dawley rats were castrated, then treated with either 1,25-D, 1,25-D with flutamide, or vehicle control. RESULTS: Incubation of primary cultures of prostate epithelial cells with 1,25-D at a concentration of 10(-8) M reduced cell proliferation by 40% of controls. The inhibition of growth by 1,25-D was maintained in the presence of DHT. Conversely, the effect of a similar dose of 1,25-D on stromal cell exposure was increased proliferation. In vivo, 1,25-D increased the prostatic weight of castrated rats that had serum testosterone levels below the detectable limit. The addition of flutamide did not alter this effect. CONCLUSIONS: These results confirm that vitamin D may be an effective antiproliferative agent of epithelial cells in prostate cancer therapy and support in vivo studies performed in the normal rat prostate.
Animals↗
Malignant priapism as the initial clinical manifestation of metastatic renal cell carcinoma with invasion of both corpora cavernosum and spongiosum.
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Aged↗
Identification of nuclear matrix protein alterations associated with renal cell carcinoma.
PURPOSE: Neoplastic transformation, including renal cell carcinoma (RCC), is always accompanied by changes in nuclear morphology. Nuclear grading of RCC is based on characteristic alterations in nuclear shape, size, area and other morphologic parameters. The nuclear matrix, which forms the skeleton of the nucleus, determines nuclear morphology. Alterations in nuclear matrix protein (NMP) composition specific to tissue and cancer type have been described in a variety of human cancers. We conducted a study to analyze the nuclear matrix protein composition of renal cell carcinoma and compare it to that of normal renal tissue and renal cell carcinoma cells grown in culture. MATERIALS AND METHODS: We analyzed the nuclear matrix protein composition of RCC tumor tissue and that of normal kidney tissue obtained from seventeen patients undergoing radical nephrectomy for RCC. We also analyzed the NMP composition of two renal cancer cell lines (A-498 and 769-P). RESULTS: We were able to identify five different and unique NMPs which were present only in the human RCC tumor samples and were absent in all normal kidney tissue. One NMP was found specifically in the normal kidney tissue. All five RCC specific NMPs were also identified in the nuclear matrix of the two cell lines analyzed. CONCLUSIONS: Five nuclear matrix proteins specific and unique to RCC were identified. These NMPs are different from those previously identified in other tissues and neoplasms. The RCC specific NMPs identified in this study can potentially be used as diagnostic markers for renal cell carcinoma and for therapeutic tumor targeting.
Aged↗
Prostatic carcinoma with signet ring cells: a clinicopathologic and immunohistochemical analysis of 12 cases, with review of the literature.
Prostatic adenocarcinoma with a signet ring cell (SRC) component is a rare, incompletely characterized variant that must be distinguished from similar tumors of bladder or gastric origin. In this study, we used mucin and immunoperoxidase stains on formalin-fixed, paraffin-embedded sections from 12 prostatic adenocarcinomas with SRC components, with antibodies to prostate-specific antigen (PSA), cytokeratins, MIB-1, bcl-2, c-MET, CD44v6, and CD44v7; we performed a comparison study on six bladder and seven gastric carcinomas with SRCs. The prostatic SRC component was always associated with the usual high-grade adenocarcinoma. Both components were positive for PSA, AE1/AE3, and CAM 5.2 (12 cases of 12) and also expressed c-MET (5 cases of 9), CD44v6 (9 of 10), and CDv7 (9 of 10). Only rare cells stained for bcl-2 (3 cases of 9). The mean MIB-1 proliferation index was 8%. Intracellular mucin was identified (periodic acid-Schiff with diastase predigestion (PAS-D) in 9 cases of 10, mucicarmine in 5 of 10, alcian blue in 6 of 10). Bladder and gastric tumors were positive for PSA (3 cases of 6 and 2 of 7, respectively), using a polyclonal antibody, and for bcl-2 (5 cases of 6, 2 of 7), c-MET (6 of 6, 6 of 7), CD44v6 (5 of 6, 6 of 7), and CD44v7 (4 of 6, 4 of 7), with mean MIB-1 proliferation indices of 15 and 35%, respectively. All were negative for cytokeratin 34 beta E12. We conclude that prostatic adenocarcinomas with SRC components are typically accompanied by high-grade adenocarcinoma; are variably positive for mucin, with PAS-D being the most sensitive stain; show expression of PSA, cytokeratins, MIB-1, bcl-2, c-MET, and CD44 similar to that shown by high-grade adenocarcinoma components; have a low MIB-1 proliferation index; and are not always distinguishable from SRC components of bladder and stomach carcinomas with any of the above stains, including PSA.
Aged↗
The biosynthesis and secretion of prostate-specific antigen in LNCaP cells.
Prostate-specific antigen (PSA) has been demonstrated to release the active form of insulin-like growth factor I in vitro (P. Cohen et al., J. Clin. Endocrinol. & Metab., 75: 1046-1053, 1992; P. Cohen et al., J. Clin. Endocrinol. & Metab., 79: 1410-1415, 1994; P. Cohen et al., Horm. Metab. Res., 26: 81-84, 1994) and has significant mitogenic activity on osteoblast cells, fibroblasts, and other cultured cells (C. S. Killian et al., Biochem. Biophys. Res. Commun., 192: 940-947, 1993). Recently, PSA has been found not only in prostate tissues but also in breast, colon, ovarian, and other tissues (E. P. Diamandis and H. Yu, J. Clin. Endocrinol. & Metab., 80: 1515-1517, 1995; E. P. Diamandis and H. Yu, Clin. Chem., 41: 204-210, 1995; A. Clements and A. Mukhtar, J. Clin. Endocrinol. & Metab., 78: 1536-1539, 1994). Therefore, PSA has been proposed as a candidate growth factor, cytokine, or growth factor regulator. In this setting, knowing how to manipulate or block the secretion of PSA by the prostate cancer cells could be a useful approach to controlling the progression of human prostate cancers. Using metabolic labeling experiments, we have studied the biosynthesis and secretion of PSA in LNCaP cells. We have also examined the effects of DTT, tunicamycin, 1-deoxymannojirimycin, pilocarpine, and testosterone on PSA biosynthesis and secretion. The results indicate that the secretion of PSA in LNCaP cells is constitutive instead of regulated and that the disruption of intramolecular disulfide bonds affects the transport of PSA from the endoplasmic reticulum to the Golgi apparatus. The biosynthesis of PSA is potentiated by testosterone and inhibited by brefeldin A and DTT. These results will help us understand PSA biosynthesis and secretion in human prostate cancers.
1-Deoxynojirimycin↗
Association between human cancer and two polymorphisms occurring together in the p21Waf1/Cip1 cyclin-dependent kinase inhibitor gene.
BACKGROUND: The cyclin-dependent kinase inhibitor gene p21Waf1/Cip1 plays a role in signaling cellular growth arrest. In response to DNA damage, p21 is induced by the p53 gene, thereby playing a direct role in mediating p53-induced G1 arrest. Alterations in this gene may adversely affect regulation of cellular proliferation and increase susceptibility for cancer. Two polymorphisms have previously been characterized in the p21 gene: a C-->A transversion at codon 31 (ser-->arg) and a C-->T transition 20 nucleotides downstream from the 3' end of exon 3. METHODS: The codon 31 polymorphism in exon 2 of the p21 gene was identified by restriction digestion (Alw26I) of products amplified by polymerase chain reaction (PCR). The polymorphism downstream from exon 3 of the p21 gene was identified by single strand conformation polymorphism (SSCP) analysis of PCR amplified products and was confirmed by PstI enzyme restriction digestion. DNA variant alleles were confirmed by direct DNA sequencing. The entire coding region and the promoter region (p53 binding domain) of the p21 gene were screened for mutations by SSCP analysis or DNA sequencing. RESULTS: The two polymorphisms were found in 18 of 96 tumor samples lacking p53 alterations (18.8%). Nine of 54 prostate adenocarcinoma samples (16.7%) contained both p21 variants, whereas 9 of 42 squamous cell carcinomas of the head and neck (21.4%) displayed both polymorphisms. Of the 110 controls examined, 10 (9.1%) had both alterations. Both p21 polymorphisms occurred together in all samples examined and there was no indication of mutation in the coding region of the p21 gene or in the p53 binding domain of the promoter region. CONCLUSIONS: These data suggest that p21 gene variants may play a role in increased susceptibility for the development of some types of cancer. In the current study, the authors demonstrated that the occurrence of these two polymorphisms is increased in prostate adenocarcinoma and squamous cell carcinoma of the head and neck. The polymorphic sites may be directly responsible for this apparent increased susceptibility or they may be linked to regulatory region alterations.
Adenocarcinoma↗
Perineural invasion in transitional cell carcinoma and the effect on prognosis following radical cystectomy.
OBJECTIVES: The relationship between perineural invasion and prognosis has been demonstrated to be poor in a number of malignancies. This has not been evaluated in the bladder. We performed a study to determine the occurrence of nodal metastases, extranodal metastases, and disease-free survival in patients with perineural invasion (PNI) and/or angiolymphatic invasion (ALI) in transitional cell carcinoma of the bladder (TCCB) from radical cystectomy specimens. METHODS: A retrospective review of 27 patients treated with radical cystectomy for TCCB was conducted. Comparisons were performed between three groups: PNI with or without ALI (PNI +/- ALI, 12 patients), ALI alone (8 patients), and a control group (no PNI or ALI) (7 patients). RESULTS: The mean patient age was 70 years (range 49 to 83). The overall median follow-up period was 11 months (range 1 to 32). PNI +/- ALI was predominantly found in Stage T3b disease (14 of 20 [70%] cases). The overall 1-year disease-free survival was 48%, 67%, and 83% for the PNI +/- ALI, ALI alone, and control groups, respectively. Nodal metastases (for all stages combined) were found in 6 of 12 (50%), 3 of 8 (38%), and 1 of 7 (14%) patients in the PNI +/- ALI, ALI alone, and control groups, respectively. Similarly, extranodal metastatic disease was found in 5 of 12 (42%), 4 of 8 (50%), and 1 of 7 (14%) patients in the PNI +/- ALI, ALI alone, and control groups, respectively. The percentage of deaths for the PNI +/- ALI, ALI only, and control groups were 33%, 50%, and 14%, respectively. CONCLUSIONS: In TCCB, perineural invasion with or without angiolymphatic invasion and angiolymphatic invasion alone are associated with a higher incidence of nodal and extranodal metastases and death.
Aged↗
A simple method for the isolation and culture of epithelial and stromal cells from benign and neoplastic prostates.
OBJECTIVES: Current primary prostate cell culture techniques use an overnight digestion or extensive media preparation. In this report, we describe a method for the culture of benign and neoplastic cells from human prostatectomy specimens that is rapid and contains no undefined factors in the medium. METHODS: Characterization of the human cultured prostate cells was performed using immunohistochemical methods and monoclonal antibodies AE1/AE3 and cytokeratin 8, as well as monoclonal antibodies against prostate-specific antigen (PSA). Polymerase chain reaction was used to measure the exclusive epithelial and stromal cell products, c-met and hepatocyte growth factor (HGF), respectively. Electron microscopy was performed to assess the cell junctions and morphologic features of epithelial cells. Optimum cell growth in different media was tested using a cell replication assay. RESULTS: Microscopic evidence revealed that the cells demonstrate typical epithelial morphology, with polyhedral cells forming tight junctions in a continuous monolayer. Desmosomes were present in electron micrographs of epithelial cells. The cultured epithelial cells described in this report also demonstrate positive cytokeratin staining. The epithelial cells reacted positively with PSA antibody, indicating that the cells retain their secretory role in cell culture for a limited period. Epithelial cells expressed the HGF receptor, c-met; stromal cells secreted HGF. Insulin, transferrin, and selenium increased the growth of cells in the chemically defined media, compared with minimum essential media (MEM) and Ham's F12. CONCLUSIONS: In summary, essentially pure cultures of prostate stromal or epithelial cells have been established using simple isolation and culture methods. These cells will be useful for the investigation of related growth factors, such as insulin-like growth factor I and insulin-like growth factor II, and in understanding the basis for stromal-epithelial cell interactions.
Cells, Cultured↗