RESUMO
The biologically active synthetic retinoid CD437 (6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene, AHPN) and different human breast carcinoma (HBC) cell lines were used to examine the possible mechanism(s) of gadd45 induction. Northern blot analysis of mRNA isolated from MCF-7, MDA-MB-468 and MDA-MB-231 HBC cell lines demonstrated a progressive increase in the 1.4 kb gadd45 transcript after exposure to 1 microM CD437. Western blot analysis showed increased gadd45 protein levels in MDA-MB-468 HBC cells following exposure to CD437. CD437 increased gadd45 mRNA levels by approximately 20-fold in MDA-MB-468 cells, however, the transcriptional activity was increased approximately 2-3-fold as demonstrated by the human gadd45 promoter-luciferase reporter construct and nuclear run-off assays. Sublines of MDA-MB-468 HBC cells expressing stably integrated GADD45 cDNA fragments were obtained and CD437-dependent induction of GADD45 analyzed. We report that approximately 300 nt located in the 5"-untranslated region (5"-UTR) of gadd45 mRNA are involved in the CD437-dependent 4-fold enhanced stability of gadd45 transcripts. MDA-MB-468 cells were stably transfected with either a plasmid having a CMV promoter-driven rabbit beta-globin gene or plasmids having a CMV promoter-driven chimeric gadd45 5"-UTR-rabbit beta-globin gene, where the entire gadd45 5"-UTR (from +1 to +298) or a 45 bp subfragment of the gadd45 5"-UTR (from +10 to +55) was positioned at the 5"-end of the rabbit beta-globin gene. CD437 was found to up-regulate expression of both the chimeric gadd45 -rabbit beta-globin transcripts, suggesting that cis element(s) involved in the CD437-dependent enhanced stability of gadd45 mRNA are contained in the 45 nt of the 5"-UTR of the gadd45 mRNA.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas/genética , Retinoides/farmacologia , Regiões 5' não Traduzidas/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Dano ao DNA/genética , Globinas/genética , Globinas/metabolismo , Meia-Vida , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Regiões Promotoras Genéticas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/genética , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Transfecção , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Proteínas GADD45RESUMO
The cyclin-dependent kinase inhibitor p21WAF1/CIP1 plays a major role in the induction of G1 cell cycle arrest following DNA damage and is known to be regulated by p53-dependent and -independent pathways. Here, we show that p21WAF1/CIP1 transcription is also regulated, independently of p53, by the cis elements that are located downstream of the transcription start site. A cDNA fragment of approximately 180 bp, located 260 bases 3' to the translation termination codon of p21WAF1/CIP1 cDNA, was cloned in both the sense and antisense orientations downstream of the CMV promoter, upstream of the SV40 promoter, and both upstream and downstream of the p21WAF1/CIP1 promoter in the plasmids carrying the luciferase reporter gene. The constructs were transiently transfected in human breast carcinoma cells MCF-7 and MDA-MB-468 and a Syrian hamster smooth muscle cell line DDT1MF2 and were found to elicit 2-3-fold or higher repression of luciferase activities. By using overlapping deletions of the above 180-bp fragment, we identified a 48-bp subfragment that contains putative cis element(s) that participate in the transcriptional repression of the p21WAF1/CIP1 gene. The overlapping subfragments bind, in vitro, to specific proteins present in the nuclear extracts of MDA-MB-468 and DDT1MF2 cells. We, therefore, propose that additional mechanism(s) exist that regulate expression of the cellular p21WAF1/CIP1 and may contribute to p21WAF1/CIP1-dependent control of the cell cycle.
Assuntos
Ciclinas/genética , DNA Complementar/genética , RNA Mensageiro/genética , Proteínas Repressoras/genética , Transcrição Gênica , Animais , Cricetinae , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Genes Reporter , Vetores Genéticos/genética , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas Nucleares , Proteínas Repressoras/fisiologia , Vírus 40 dos Símios/genética , TransfecçãoRESUMO
Trans retinoic acid (RA) has proven to be a potent therapeutic agent in the treatment of acute promyelocytic leukemia. Unfortunately, other subtypes of acute myelogenous leukemia are resistant to the antiproliferative and differentiating effects of RA. In this report, we describe a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN; CD437) that not only totally inhibits the proliferation of RA-resistant leukemic cell lines HL-60R and K562 but also induces apoptosis in these cells. Exposure of HL-60R to CD437 results in the rapid (within 30 minutes) increase of the cyclin-dependent kinase inhibitor p21(waf1/cip1) as well as GADD45 mRNA. Manifestations of CD437-mediated programmed cell death are noted within 2 hours, as indicated by both the cleavage and activation of the CPP32 protease and cleavage of poly (ADP-ribose) polymerase. This is followed by cleavage of bcl-2 and internucleosomal DNA degradation. HL-60R cells do not express the retinoid nuclear receptor RAR beta and RAR gamma and express a truncated RAR alpha. Thus, CD437 induction of p21(waf1/cip1) and GADD45 mRNAs and apoptosis occurs through a unique mechanism not involving the retinoid nuclear receptors. CD437 represents a unique retinoid with therapeutic potential in the treatment of myeloid leukemia.
Assuntos
Apoptose/efeitos dos fármacos , Caspases , Células HL-60/efeitos dos fármacos , Naftalenos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Retinoides/farmacologia , Antineoplásicos/farmacologia , Caspase 3 , Diferenciação Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , Cisteína Endopeptidases/metabolismo , Fragmentação do DNA , Resistencia a Medicamentos Antineoplásicos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Biossíntese de Proteínas , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Ácido Retinoico/deficiência , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Tretinoína/farmacologia , Células Tumorais Cultivadas , Proteínas GADD45 , Receptor gama de Ácido RetinoicoRESUMO
Estradiol-mediated enhancement of retinoic acid receptor alpha (RARalpha) expression in the estrogen receptor (ER)-positive human breast carcinoma (HBC) cells results in their sensitivity to RA-mediated growth inhibition (A. K. Rishi et al., Cancer Res., 55: 4999-5006, 1995). Most ER-negative HBCs are known to express lower levels of RARalpha and are resistant to RA-mediated inhibition of growth. We show that ER-negative SKBR-3 and MDA-MB-435 HBCs express approximately 2-fold higher levels of RARalpha isoform 1 mRNA when compared to the ER-negative MDA-MB-231 and MDA-MB-468 HBCs. SKBR-3 cells are sensitive to growth inhibition by RA, and by using RARalpha-selective synthetic retinoids, we demonstrate that the antiproliferative effects of RA in the SKBR-3 cell line are accomplished, in part, via activation of RARalpha. Both MDA-MB-231 and MDA-MB-468 HBCs are not growth inhibited by RA or any of the retinoids tested. Transient transfection experiments using a 5.0-kb RARalpha promoter fragment fused to the luciferase reporter gene showed 2-3-fold higher transcriptional activation in SKBR-3 cells when compared to MDA-MB-468 cells. We report identification of a 72-bp fragment of RARalpha promoter that contains unique cis elements responsible for mediating an estradiol-independent 2.5-fold enhancement of RARalpha gene expression in SKBR-3 and MDA-MB-435 cells.
