p53 was sequenced in tumor samples from cohort 1 using either genomic DNA or cDNA as a tem plate with primers derived from intronic or gene specific sequences. PCR amplification and purifica tion was performed as previously described Vismodegib IC50 and sequenced at Eurofins MWG Operon. Cohort 2, Breast cancer patients were diagnosed and operated on between 1987 and 1989 at Uppsala University Hospital, Uppsala, Sweden. The median age for the patients used in this study was 63 years. Clinicopathological data and treatment have been previously reported. Briefly, patients were operated on and received postoperative radiotherapy. When adjuvant tamoxifen was given, some patients received this treatment as part of a randomized study comparing two versus five years, and chemotherapy, mostly CMF according to standards in those days.

Ethical permission was obtained from the ethical committee at Karolinska Institute and with informed consent from the patients. Data on p53 mutational sta tus in Inhibitors,Modulators,Libraries this series of breast tumor specimens have been described. Genomic DNA and RNA from fresh frozen tumor tis sue were isolated as previously described. RNA from various normal tissues was purchased from ABI. Normal breast tissue was obtained from reduction surgeries as well as from noncancerous tissue DNA extracted from paraffin embedded breast cancer specimens. cDNA was synthesized from 2 ug of total RNA using Superscript First strand Synthesis System. Cell lines, transfections and treatments A total of 60 human Inhibitors,Modulators,Libraries cancer cell lines, originating from breast, brain, prostate, kidney, blood, cervix, lung, skin, bone and thyroid, were analyzed for FBXW7 hCDC4 expression and promoter methylation.

Cell lines were maintained and cultured according to American Type Culture Collection guidelines or as previously Inhibitors,Modulators,Libraries described. All plasmid transfections were performed using LT1 transfection reagent, as recommended by the manufacturers protocol. For experiments evaluat ing the effects of demethylation, Inhibitors,Modulators,Libraries cell lines maintained in appropriate media were treated with 5 aza 2 deoxycyto dine or DMSO for three to five days. Each promoter region was subcloned into pGL3 vector after KpnI and BglII restriction digestion. The result ing promoter constructs were termed pGL3hCDC4b 1. 6, pGL3hCDC4b 0. 8. and pGL3hCDC4b 0. 6. The pRL SV40 Renilla luciferase plasmid was obtained from Pro mega.

PCR reactions were performed in a BioRad thermocycler. Inhibitors,Modulators,Libraries Luciferase assay Luciferase GW 572016 activities in cell lysates from cells transfected with different pGL3 constructs and pRL SV40 control plasmid were measured in a luminometer using the Dual Luciferase Reporter Assay System according to the man ufacturers protocol. Luciferase activities were quantified and fold change was averaged from at least three separate experiments performed in triplicates.