Neoplasms are commonly characterized by abnormal gene expression. Our studies will utilize a unique murine pituitary tumor model (MGH-101A) to investigate the relationship between altered thyroid hormone receptor (TR) gene activity and malignant transformation. This tumor evolved in our laboratory from a thyrotropic tumor. It is unique in that it underwent malignant transformation in association with altered responses to thyroid hormone. Specifically, cellular growth and alpha-subunit production, which are normally tightly regulated by thyroid hormone in this cellular phenotype, have become non-regulated and promiscuous. Our preliminary observations suggest that these defects occur because of an abnormality in transcription of the beta form of the TR gene. The projected studies in this pituitary neoplasm will be divided into three parts. First, we will investigate the molecular basis for absent transcription of the beta form of the TR gene in MGH-101A by delineating its structure and organization. Second, we will determine the functional integrity of the TRbeta1/beta2 gene promoter(s) in MGH-101A compared to authentic thyrotropic cells. The production of the TRbeta2 is normally a highly tissue-specific function in pituitary cells. Its absence therefore could be related to an abnormality in the TRbeta gene structure or the presence or absence of trans-acting factors altering expression of this gene. We will use techniques of molecular biology to discover the mechanisms for the absent TRbeta2 gene expression. A solution to this problem could provide insights into similar phenomenon where normal cellular development and thyroid hormone action are linked. The coupling of differentiated cellular function and thyroid hormone action may have broader implications for normal and abnormal cellular biology. Third, we will reconstitute normal TRbeta1/beta2 isotypes in MGH-101A and study their effects on cellular growth and alpha-subunit regulation of thyroid hormone. For these experiments we will utilize our recently cloned permanent cell line of MGH-101A to produce transient and stable transfectants expressing TRbeta1 and TRbeta2 cDNAs. We will evaluate whether normal regulation of alpha-subunit production at the transcriptional level is reconstituted by this procedure by analyzing alpha-subunit gene transcription, alpha-subunit protein production, and TR isoform distribution. Furthermore, utilizing stable transfectants which express the TRbeta2 cDNA, we will determine in vitro and in vivo whether growth of these cells is now normally regulated by thyroid hormone. Knowledge gained on thyroid hormone regulation of this tumor's growth and alpha-subunit production will contribute substantive information on thyroid hormone action as it relates to differentiated and undifferentiated cellular functions.
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