Activation of PTHrP gene expression in squamous carcinoma cell lines by mutant isoforms of the tumor suppressor p53

We have evaluated the status of p53 expression in three squamous carcinoma cell fines that express high levels of PTHrP mRNA and protein and also caus e hypercalcemia when grown in nude mice. All three of these lines possess a single p53 allele, each of which harbors a missense point mutation that gi ves rise to a mutant p53 protein with a denatured conformation. Using site- directed mutagenesis, we created a p53 expression construct hearing a misse nse mutation at codon 158, identical to that expressed by one of the cell l ines. This construct and p53 constructs expressing representative denatured conformation mutants were then used to develop stably transfected lines, w hich expressed increased levels of PTHrP mRNA. Promoter-specific RNase prot ection indicated that this increase was due primarily to transcripts origin ating from the two TATA promoters, and not the GC-rich initiator element wi thin the PTHrP gene. Cotransfection of mutant p53 expression vectors with a series of reporter constructs under the control of the human PTHrP promote r region showed that mutant p53 isoforms activated constructs containing mu ltiple promoter elements and flanking sequences, but failed to activate con structs with individual promoters in isolation. These findings suggest that the activation of PTHrP gene expression by mutant p53 isoforms displaying a denatured conformation is dependent on interactions with sequences in the PTHrP gene regulatory region beyond the basal TATA promoters.