Abstract
BackgroundProlactin 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.MethodsRabbits 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.ResultsWe 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.ConclusionsDifferences 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.
Highlights
Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland
With the discovery of the various isoforms of the prolactin receptor (PRLR), a more detailed analysis of the cellular localization of the receptor as well as possible differences between subtypes of breast cancer was warranted. To facilitate these studies we developed and characterized PRLR isoform specific polyclonal antibodies that reveal that three isoforms, LF, SF1a and SF1b, are differentially expressed in ductal and lobular carcinoma tissues
CHO cells were transiently transfected with cDNA containing the individual PRLR isoforms; Figure. 1A shows the specificity of our antibodies by western blot
Summary
Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. 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. The role of prolactin (PRL) in human breast cancer is becoming more clearly defined. There are five cell-associated isoforms of the human PRLR, long (LF), intermediate, ΔS1, and two short forms (SF1a and SF1b) [4,9] that differ only in their C-terminal cytoplasmic domains. Studies from our laboratory and from others [7,12] have demonstrated that mRNA for the three specific isoforms of the PRLR is expressed in both normal and cancerous human breast cells and tissues
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