Novel role of HDAC inhibitors in AML1/ETO AML cells: activation of apoptosis and phagocytosis through induction of annexin A1.

The chimeric fusion protein AML1-ETO, created by the t(8;21) translocation, recruits histone deacetylase (HDAC) to AML1-dependent promoters, resulting in transcriptional repression of the target genes. We analyzed the transcriptional changes in t(8;21) Kasumi-1 AML cells in response to the HDAC inhibitors, depsipeptide (FK228) and suberoylanilide hydroxamic acid (SAHA), which induced marked growth inhibition and apoptosis. Using cDNA array, annexin A1 (ANXA1) was identified as one of the FK228-induced genes. Induction of ANXA1 mRNA was associated with histone acetylation in ANXA1 promoter and reversal of the HDAC-dependent suppression of C/EBPalpha by AML1-ETO with direct recruitment of C/EBPalpha to ANXA1 promoter. This led to increase in the N-terminal cleaved isoform of ANXA1 protein and accumulation of ANXA1 on cell membrane. Neutralization with anti-ANXA1 antibody or gene silencing with ANXA1 siRNA inhibited FK228-induced apoptosis, suggesting that the upregulation of endogenous ANXA1 promotes cell death. FK228-induced ANXA1 expression was associated with massive increase in cell attachment and engulfment of Kasumi-1 cells by human THP-1-derived macrophages, which was completely abrogated with ANXA1 knockdown via siRNA transfection or ANXA1 neutralization. These findings identify a novel mechanism of action of HDAC inhibitors, which induce the expression and externalization of ANXA1 in leukemic cells, which in turn mediates the phagocytic clearance of apoptotic cells by macrophages.

The novel triterpenoid CDDO-Me suppresses MAPK pathways and promotes p38 activation in acute myeloid leukemia cells.

Development of novel therapeutic strategies is a continuing challenge for the treatment of acute myeloid leukemia (AML). The novel triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), induces apoptosis in myeloid leukemic cell lines and in primary AML samples. In this report, the effects of CDDO-Me on CD34(+) AML progenitor cells in vitro were examined. CDDO-Me induced apoptosis in all but one of ten AML samples. CDDO-Me is known to inhibit the activation of ERK1/2. In this series of primary AML samples, ERK was expressed and phosphorylated in all patient samples studied and CDDO-Me inhibited ERK phosphorylation in five of 10 samples. However, CDDO-Me induced apoptosis in four of five samples without decreasing pERK levels, suggesting that pERK is not the sole target of the compound. CDDO-Me induced phosphorylation of p38 in AML-derived U937 cells. Pretreatment of U937 cells with a p38 inhibitor protected cells from the cyto-toxic effects of CDDO-Me. These findings suggest a role for p38 in CDDO-Me-induced apoptosis. In preliminary studies, CDDO-Me induced p38 phosphorylation in seven of eight primary AML samples. These findings suggest that CDDO-Me treatment shifts cell signaling away from cyto-protective pathways and thus CDDO-Me may be effective for the treatment of AML.

Ki67 expression levels are a better marker of reduced melanoma growth following MEK inhibitor treatment than phospho-ERK levels.

The loss of tumour phospho-extracellular responsive kinase (pERK) positivity is the major treatment biomarker for mitogen-activated protein kinase/extracellular responsive kinase (MEK) inhibitors. Here, we demonstrate that there is a poor correlation between pERK inhibition and the anti-proliferative effects of MEK inhibitors in melanoma cells. We suggest that Ki67 is a better biomarker for future clinical studies.

HPV16/18 prevalence in cervical lesions/cancers and p53 genotypes in cervical cancer patients from India.

OBJECTIVES: The HPV16/18 code for an oncoprotein-E6, which binds to p53 tumor suppressor protein and degrades the protein via ubiquitination. A common polymorphism of p53 in exon 4 codon 72, resulting in either proline (Pro) or arginine (Arg), affects HPV16/18 E6-mediated degradation of p53 protein in vivo. Hence, in the current study we investigated the prevalence of HPV16/18 in cervical lesions and the distribution of p53 genotypes in cervical cancers and normal healthy women. METHODS: DNA from 337 Indian women with invasive cervical cancers, 164 women with clinically normal cervix, 64 women with low-grade squamous intraepithelial lesions (LSIL), and 5 women with high-grade squamous intraepithelial lesions (HSIL) was examined for the presence of HPV16/18 using consensus primers in a polymerase chain reaction (PCR), and the specific HPV type was identified by Southern hybridization of the PCR product using HPV16/18 type-specific nucleotide sequences as probes. Further, 134 women with cervical cancers and 131 healthy women were used to determine the frequency of p53 genotypes, Pro/Pro, Arg/Arg, and Pro/Arg, using peripheral blood cell DNA to indicate the constitutional genotypes and allele-specific primers, in a PCR-based assay. RESULTS: We observed a prevalence of HPV16/18 in 77% (258/337) of cervical cancer patients, 38% (24/64) of LSILs, 4 of 5 HSILs, and 15.2% (25/164) of normal healthy women. The frequency of distribution of the three genotypes of p53 codon 72 in a subgroup of the HPV16/18-positive cervical cancer patients was Pro/Pro 0.18 and Arg/Arg 0.26, with the heterozygous Pro/Arg 0.56, differing significantly from the genotype frequency in the normal healthy women (chi(2) = 6.928, df = 2, P < 0.05). CONCLUSIONS: A high prevalence of HPV16/18 was observed in the cervical cancers. The prevalence in LSILs confirms HPV16/18 infection as an early event and further indicates a role in progression of lesions. The p53 genotype distribution indicated that women homozygous for Arg genotype were at a 2.4-fold higher risk for developing HPV16/18-associated cervical carcinomas, compared to those showing heterozygous Pro/Arg genotype (odds ratio 2.4, 95% confidence interval 1.89 to 3.04).