Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies.

BACKGROUND: Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR) are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. METHODS: Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. RESULTS: We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76%) of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72%) and 27% had only low levels of expression. CONCLUSIONS: Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in treatment of this disease by providing new reagents to study the protein expression of the human PRLR.

Increases in apoptosis and declines in Bcl-XL protein characterise testicular regression in American crows (Corvus brachyrhynchos).

The present study investigated the cellular changes observed during testicular regression in American crows. Testes from adults caught during the early (March), progressing (April), peak (early May), transitional (late May), and post- (June) breeding season were examined. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and Bcl-X(L) protein immunolabelling. Testis mass increased two-fold from March to early May (P < 0.05), then declined 19-fold by June (P < 0.001) without corresponding changes in body mass (P > 0.05). Testicular activity, evaluated using a spermatogenic index, increased nearly two-fold from March to early May and declined nine-fold in June (P < 0.001). Seminiferous tubule diameter declined four-fold in June compared with earlier months (P < 0.001). In all testes, TUNEL-positive germ cells were detected at low levels, with the highest levels observed in late May (P < 0.001). In contrast, TUNEL-positive Sertoli cells were maintained at low levels in March-April and increased nine-fold in early May (P < 0.001). The Bcl-X(L) immunostaining was detected in Sertoli cells in March-early May; however, staining was most intense in March-April and substantially weaker by early May. These data suggest that the seasonal rise in testicular competence occurs slowly in American crows; however, testis function is terminated rapidly after the breeding season. Furthermore, it is likely that Sertoli cell apoptosis followed by massive germ cell loss is responsible for the rapid reduction in testis mass.