Koeffler Hp

p53 transactivation through various p53-responsive elements

p53 is a nuclear phosphoprotein whose function is classified as tumor suppression. Studies have shown that p53 functions by binding to p53 DNA recognition sequences and regulates transcription of growth‐regulatory genes. Various p53 recognition sequences have recently been identified. pOST2 contained two copies of a palindromic high‐affinity DNA‐binding sequence for p53; the other p53 recognition sequences included p53‐binding fragments found in the human ribosomal gene cluster (pRGC) region and in the murine muscle creatine kinase promoter (pMCK). The purpose of this study was to compare the abilities of various p53 recognition sequences to mediate transcription in the presence of endogenously produced wild‐type (wt) or mutant p53. Three p53‐responsive chloramphenicol acetyltransferase (CAT) reporter constructs (pOST2, pRGC, and pMCK) that contain one or two copies of p53 recognition sequences upstream of a herpes thymidine kinase (TK) promoter and CAT reporter cDNA were constructed. Either a p53‐responsive gene or a control reporter gene was transfected into human carcinoma cell lines (having various p53 mutations) either with or without a wt or mutant p53 expression vector. CAT activity was assayed to measure transactivation through the various p53‐responsive elements. We showed that pOST2 had a greater ability to mediate transactivation by p53 than either pRGC or pMCK. p53 with a mutation at either codon 175 or 248 was unable to transactivate a reporter gene with pOST2, pRGC, or pMCK. We found it interesting that pOST2, but not pRGC or pMCK, was able to mediate transactivation in cell lines that produce codon 273‐mutant p53. These findings suggest that the various sensitivities of the different p53‐responsive elements to specific mutant and wt p53s may be an important factor in the role of p53 as a transcriptional activator both under normal physiological conditions and during carcinogenesis.

Marked Effect of the Combination of a Novel Vitamin D3 Analog, KH1060, and 9-cis-RA on Inhibition of Clonal Growth, Decreases of bcl-2 Level and Induction of Apoptosis in HL-60 Cells

All-trans retinoic acid (RA) is the first highly effective differentiation-inducing agent for remission induction in patients with APL, but remissions are short-lived because the treatment fails to eliminate the malignant clone and clinical resistance develops rapidly. Another retinoic acid compound, 9-cis-RA, unlike alltrans-RA—which binds only retinoic acid receptors (RARs)—is a high affinity ligand for both RARs and RXRs. To prevent or overcome resistance and to eliminate the malignant clone, in analogy with chemotherapy, combinations of new potent differentiation-inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 [1,25 dihydroxyvitamin D3 (1, 25D)] is an inhibitor of proliferation and effector of differentiation of myeloid leukemic cells to monocytes. The 20-epi-22oxa-24a, 26a, 27a-tri-homo-1α,25(OH)2D3 (KH 1060) belongs to the family of 20-epi-vitamin D3 analogs and is considerably more potent in vitro than 1,25D as a regulator of growth of the HL-60 cell line. The aim of this study was to evaluate the affect of the combination of KH 1060 with 9-cis-RA on proliferation of the human promyelocytic leukemia cell line HL-60. The 9-cis-RA (10-8M) produced a 30% inhibition of clonal proliferation of HL-60, and KH 1060 at 10-9 M resulted in 50% inhibition of clonal proliferation of the leukemic cells. No colonies were detectable after incubation with 10-7 MKH 1060. The combination of 9-cis-RA (10-8M) with KH 1060 (10-9M) produced a 95% inhibition of clonal growth. When the HL-60 clonogenic cells were cultured in liquid medium with either 10-7 M of KH 1060 or 9-cis-RA for 3 days, washed and plated in soft agar, KH 1060 and 9-cis-RA inhibited 54% and 30% of the HL-60 cells, respectively. In constrast, when the cells were cultured in liquid culture with 10-7 M KH 1060 and 9-cis-RA together for 3 days, washed and plated in soft agar, no colonies were detectable. In order to gain insight into the remarkable antileukemic effect of the combination of these analogs, apoptosis and expression of bcl-2 were examined. After 3 days of culture of HL-60 cells with the combination of KH 1060 (10-7 M) and 9-cis-RA (107 M), apoptosis was induced in 62% of cells, as detected by measurement of morphological changes. Also after 3 days, bcl-2 protein, as analyzed by immuno-histochemistry, became nearly undetectable when cells were cultured with both KH 1060 and 9-cis-RA (10-7 M). When cultured over the same period with either KH 1060 or 9-cis-RA (107 M), 26% and 36% of cells expressed bcl-2, respectively, and 100% of wild-type HL-60 expressed bcl-2. In summary, our data demonstrate that the combination of KH 1060 and 9-cis-RA ireversibly and synergistically inhibited clonal growth and induced apoptosis of HL-60 cells concomitantly with a very marked decrease expression of bcl-2.