Androgen receptor CAG repeat polymorphisms in canine prostate cancer.

BACKGROUND: Relatively shorter lengths of the polymorphic polyglutamine repeat-1 of the androgen receptor (AR) have been associated with an increased risk of prostate cancer (PC) in humans. In the dog, there are 2 polymorphic CAG repeat (CAGr) regions. OBJECTIVE: To investigate the relationship of CAGr length of the canine AR-gene and the development of PC. ANIMALS: Thirty-two dogs with PC and 172 control dogs were used. METHODS: DNA was extracted from blood. Both CAG repeats were amplified by polymerase chain reaction (PCR) and PCR products were sequenced. RESULTS: In dogs with PC, CAG-1 repeat length was shorter (P = .001) by an increased proportion of 10 repeats (P = .011) and no 12 repeats (P = .0017) than in the control dogs. No significant changes were found in CAG-3 length distribution. CAG-1 and CAG-3 polymorphisms proved not to be in linkage disequilibrium. Breed difference in allelic distribution was found in the control group. Of the prostate-disease sensitive breeds, a high percentage (64.5%) of the shortest haplotype 10/11 was found in the Doberman, whereas Beagles and German Pointers had higher haplotype 12/11 (47.1 and 50%). Bernese Mountain dogs and Bouvier dogs both shared a high percentage of 11 CAG-1 repeats and 13 CAG-3 repeats. Differences in (combined) allelic distributions among breeds were not significant. CONCLUSIONS AND CLINICAL IMPORTANCE: In this preliminary study, short CAG-1 repeats in the AR-gene were associated with an increased risk of developing canine PC. Although breed-specific differences in allelic distribution of CAG-1 and CAG-3 repeats were found, these could not be related to PC risk.

cDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype.

Mammary cancer is the most common type of cancer in female dogs with a lifetime risk of over 24% when dogs are not spayed. The elucidation of the complete canine genome opens new areas for development of cancer therapies. These should be tested first by in vitro models such as cell lines. However, to date, no canine mammary cell lines have been characterized by expression profiling. In this study, canine mammary tumour cell lines with histologically distinct primary tumours of origin were characterized using a newly developed canine cDNA microarray. Comparisons of gene expression profiles showed enrichment for distinct biological pathways and were related to biological properties of the cell lines such as growth rate and in vitro tumourigenicity. Additionally, gene expression profiles of cell lines also showed correspondence to their tumour of origin. Major differences were found in Wnt, cell cycle, cytokine/Rho-GTPase, alternative complement and integrin signalling pathways. Because these pathways show an overlap at the molecular level with those found in human breast cancer, the expression profiling of spontaneous canine mammary cancer may also function as a biological sieve to identify conserved gene expression or pathway profiles of evolutionary significance that are involved in tumourigenesis. These results are the basis for further characterization of canine mammary carcinomas and development of new therapies directed towards specific pathways. In addition these cell lines can be used to further investigate identified deregulated pathways and characterize until now unannotated genes.

Expression of the scaffolding subunit A of protein phosphatase 2A during rat testicular development.

Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A (PP2A-A) is the major subunit in the formation of a functional PP2A holoenzyme. In this study, we investigated the expression of PP2A-A during testicular development in more detail using in situ hybridization, immunohistochemistry, and Western blot with testes of rats of various ages from 16 days postcoitum (pc) to adulthood. The expression of PP2A-A was detected in fetal proliferative gonocytes at 16 days pc, declining thereafter during the quiescent period of the gonocytes. From the day of birth to the start of spermatogenesis (Day 4 postpartum [pp]), the number of PP2A-A-immunopositive gonocytes increased. At Day 4 pp, the first A1 spermatogonia appeared along the basement membrane; all were PP2A-A positive. In the adult, PP2A-A was upregulated during the differentiation of the As, Apr, and Aal spermatogonia to the A1 spermatogonia and expressed thereafter by all other spermatogonia. Spermatocytes from the pachytene stage onward and all spermatids in the adult testis also showed clear expression of PP2A-A. In Sertoli cells, PP2A-A was detected during their proliferative period at 19 days pc to 15 days pp. The presence of a functional enzyme was confirmed by the additional detection of the catalytic subunit C of PP2A using Western blot analyses at various ages during testicular development. This apparent pattern of expression of PP2A-A during testicular development suggests that PP2A may play an important role in the proliferation of distinct populations of testicular cells and during meiosis and sperm maturation.

Immunomagnetic isolation of fetal rat gonocytes.

PROBLEM: An efficient method to obtain highly enriched populations of viable gonocytes from rat embryos at Day 18 and Day 20 postcoitum (pc) is described. METHOD: Single-cell suspensions with high cell yield were obtained by a collagenase/ trypsin digestion of the decapsulated testis. The gonocytes were purified by a direct immunoseparation technique, using magnetizable beads coated with rat anti-mouse immunoglobulin M (IgM) and a monoclonal antibody 4B6.3E10, which specifically reacted with a differentiation antigen on the fetal germ cells. RESULTS: Populations of 8.3 +/- 2.7 (x10(3); 18 days pc) or 1.2 +/- 0.25 (x10(4); 20 days pc) viable gonocytes per testis with purities of 91 +/- 6.5% and 92 +/- 4.3%, respectively, as determined by Nomarski microscopy were obtained. CONCLUSION: The cells were successfully used for culture studies and as starting material for the investigation of gene expression.