Endocrine Abstracts

Parathyroid hormone-related peptide (PTHrP)have been found to be expressed in a variety of human tumors including colon and it was originally identified as the causative agent of humoral hypercalcemia of malignancy (HHM), We used a human colonic cell line (LOVO) as a model to study the mechanism for the proliferative effects of PTHrP. The PTHrP constructs expressing PTHrP in sense or antisense orientation were constructed by cloning hPTHrP complementary DNA (cDNA) coding for amino acids -5 to +139 in the expression vectors pCDNA3 (+P) or (-P), respectively. The (+P) and (-P) refer to the orientation of the multiple cloning site within the vector, relative to the direction of transcription from the T7 promoter. These constructs, as well as pCDNA3 (V) as the empty vector control, were transfected into these cells. Two days after transfection, 100 ug/ml G418 was added, and resistant clones were selected. Single clones of stably transfected cells was isolated and plated in 24-well plates. Cells were then grown in F-12 medium and 10% fetal bovine serum (FBS) and incubated for 2, 4 and 7 days. The proliferation rate of LOVO clones (+P) overexpressing PTHrP was less rapid than that of empty vector transfected cells, however, at day 7, the cell number of the PTHrP overexpressing clones (-P) was more than two folds compared with the empty vector transfected clones (V). In conclusion, this data may leads to significant therapeutic implications for the role of PTHrP in colon cancer.