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ZNF750 controls epithelial homeostasis by regulating epidermal-differentiation genes, a role underscored by its pathogenic mutations in esophageal squamous cell cancers (SCCs). However, the precise role of ZNF750 in SCC cell biology remains unclear. In this study, we report that ZNF750 is exclusively deleted, mutated and underexpressed in human SCCs, and low ZNF750 expression is associated with poor survival. Restoration of wildtype, but not mutant ZNF750 protein uniquely inhibited the malignant phenotypes of SCC cells both in vitro and in vivo. Notably, ZNF750 promoted the expression of a long non-coding RNA (TINCR), which mediated both cancer-inhibition and differentiation-induction effects of ZNF750. In addition, ZNF750 potently suppressed cell migration by directly inhibiting the transactivation of LAMC2. Together, our findings characterize ZNF750 as a crucial SCC-specific suppressor and uncover its novel anticancer-associated functions.

Definition of preleukemia has evolved. It was first used to describe the myelodysplastic syndrome (MDS) with a propensity to progress to acute myeloid leukemia (AML). Individuals with germline mutations of either RUNX1, CEBPA, or GATA2 can also be called as preleukemic because they have a markedly increased incidence of evolution into AML. Also, alkylating chemotherapy or radiation can cause MDS/preleukemia, which nearly always progress to AML. More recently, investigators noted that AML patients who achieved complete morphological remission after chemotherapy often have clonal hematopoiesis predominantly marked by either DNMT3A, TET2 or IDH1/2 mutations, which were also present at diagnosis of AML. This preleukemic clone represents involvement of an early hematopoietic stem cells, which is resistant to standard therapy. The same clonal hematopoietic mutations have been identified in older 'normal' individuals who have a modest increased risk of developing frank AML. These individuals have occasionally been said, probably inappropriately, to have a preleukemia clone. Our evolving understanding of the term preleukemia has occurred by advancing technology including studies of X chromosome inactivation, cytogenetics and more recently deep nucleotide sequencing.

Long non-coding RNAs (lncRNAs) have a critical role in cancer initiation and progression, and thus may mediate oncogenic or tumor suppressing effects, as well as be a new class of cancer therapeutic targets. We performed high-throughput sequencing of RNA (RNA-seq) to investigate the expression level of lncRNAs and protein-coding genes in 30 esophageal samples, comprised of 15 esophageal squamous cell carcinoma (ESCC) samples and their 15 paired non-tumor tissues. We further developed an integrative bioinformatics method, denoted URW-LPE, to identify key functional lncRNAs that regulate expression of downstream protein-coding genes in ESCC. A number of known onco-lncRNA and many putative novel ones were effectively identified by URW-LPE. Importantly, we identified lncRNA625 as a novel regulator of ESCC cell proliferation, invasion and migration. ESCC patients with high lncRNA625 expression had significantly shorter survival time than those with low expression. LncRNA625 also showed specific prognostic value for patients with metastatic ESCC. Finally, we identified E1A-binding protein p300 (EP300) as a downstream executor of lncRNA625-induced transcriptional responses. These findings establish a catalog of novel cancer-associated functional lncRNAs, which will promote our understanding of lncRNA-mediated regulation in this malignancy.

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes.

Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation ofoncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation ofErk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels,we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse modelsfor pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance. Shojaee etal. show that successful transformation of pre-B cells to pre-B acute lymphoblastic leukemia (ALL) requires negative feedback regulation of Erk signaling and inhibiting this feedback selectively kills pre-B ALL cells, suggesting negative feedback regulation of oncogenes as a vulnerability in human ALL.

LNK (SH2B3) is an adaptor protein studied extensively in normal and malignant hematopoietic cells. In these cells, it downregulates activated tyrosine kinases at the cell surface resulting in an antiproliferative effect. To date, no studies have examined activities of LNK in solid tumors. In this study, we found by in silico analysis and staining tissue arrays that the levels of LNK expression were elevated in high-grade ovarian cancer. To test the functional importance of this observation, LNK was either overexpressed or silenced in several ovarian cancer cell lines. Remarkably, overexpression of LNK rendered the cells resistant to death induced by either serum starvation or nutrient deprivation, and generated larger tumors using a murine xenograft model. In contrast, silencing of LNK decreased ovarian cancer cell growth in vitro and in vivo. Western blot studies indicated that overexpression of LNK upregulated and extended the transduction of the mitogenic signal, whereas silencing of LNK produced the opposite effects. Furthermore, forced expression of LNK reduced cell size, inhibited cell migration and markedly enhanced cell adhesion. Liquid chromatography-mass spectroscopy identified 14-3-3 as one of the LNK-binding partners. Our results suggest that in contrast to the findings in hematologic malignancies, the adaptor protein LNK acts as a positive signal transduction modulator in ovarian cancers.

BackgroundWe investigated the biologic and pharmacologic activities of a chromosome region maintenance 1 (CRM1) inhibitor against human non-small cell lung cancer (NSCLC) cells both in vitro and in vivo.MethodsThe in vitro and in vivo effects of a novel CRM1 inhibitor (KPT-330) for a large number of anticancer parameters were evaluated using a large panel of 11 NSCLC cell lines containing different key driver mutations. Mice bearing human NSCLC xenografts were treated with KPT-330, and tumour growth was assessed.ResultsKPT-330 inhibited proliferation and induced cell cycle arrest and apoptosis-related proteins in 11 NSCLC cells lines. Moreover, the combination of KPT-330 with cisplatin synergistically enhanced the cell kill of the NSCLC cells in vitro. Human NSCLC tumours growing in immunodeficient mice were markedly inhibited by KPT-330. Also, KPT-330 was effective even against NSCLC cells with a transforming mutation of either exon 20 of EGFR, TP53, phosphatase and tensin homologue, RAS or PIK3CA, suggesting the drug might be effective against a variety of lung cancers irrespective of their driver mutation.ConclusionsOur results support clinical testing of KPT-330 as a novel therapeutic strategy for NSCLC.

High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0-12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P

BackgroundThe PIAS4 protein belongs to the family of protein inhibitors of activated STAT, but has since been implicated in various biological activities including the post-translational modification known as sumoylation. In this study, we explored the roles of PIAS4 in pancreatic tumourigenesis.MethodsThe expression levels of PIAS4 in pancreatic cancer cells were examined. Cell proliferation and invasion was studied after overexpression and gene silencing of PIAS4. The effect of PIAS4 on hypoxia signalling was investigated.ResultsThe protein was overexpressed in pancreatic cancer cells compared with the normal pancreas. Gene silencing by PIAS4 small interfering RNA (siRNA) suppressed pancreatic cancer cell growth and overexpression of PIAS4 induced expression of genes related to cell growth. The overexpression of PIAS4 is essential for the regulation of the hypoxia signalling pathway. PIAS4 interacts with the tumour suppressor von Hippel-Lindau (VHL) and leads to VHL sumoylation, oligomerization, and impaired function. Pancreatic cancer cells (Panc0327, MiaPaCa2) treated with PIAS4 siRNA suppressed expression of the hypoxia-inducible factor hypoxia-inducible factor 1 alpha and its target genes JMJD1A, VEGF, and STAT3.ConclusionOur study elucidates the role of PIAS4 in the regulation of pancreatic cancer cell growth, where the suppression of its activity represents a novel therapeutic target for pancreatic cancers.

Approximately 90% of well-differentiated/de-differentiated liposarcomas (WDLPS/DDLPS), the most common LPS subtype, have chromosomal amplification at 12q13-q22. Many protein-coding genes in the region, such as MDM2 and , have been studied as potential therapeutic targets for LPS treatment, with minimal success. In the amplified region near the MDM2 gene, our single nucleotide polymorphism (SNP) array analysis of 75 LPS samples identified frequent amplification of miR-26a-2. Besides being in the amplicon, miR-26a-2 was overexpressed significantly in WDLPS/DDLPS (P

PAX5 encodes a master regulator of B-cell development. It fuses to other genes associated with acute lymphoblastoid leukemia (ALL). These fusion products are potent dominant-negative (DN) inhibitors of wild-type PAX5, resulting in a blockade of B-cell differentiation. Here, we show that multimerization of PAX5 DNA-binding domain (DBD) is necessary and sufficient to cause extremely stable chromatin binding and DN activity. ALL-associated PAX5-C20S results from fusion of the N-terminal region of PAX5, including its paired DBD, to the C-terminus of C20orf112, a protein of unknown function. We report that PAX5-C20S is a tetramer, which interacts extraordinarily stably with chromatin as determined by Fluorescence Recovery After Photobleaching in living cells. Tetramerization, stable chromatin binding and DN activity all require a putative five-turn amphipathic α-helix at the C-terminus of C20orf112, and does not require potential corepressor binding peptides elsewhere in the sequence. In vitro, the monomeric PAX5 DBD and PAX5-C20S binds a PAX5-binding site with equal affinity when it is at the center of an oligonucleotide too short to bind to more than one PAX5 DBD. But, PAX5-C20S binds the same sequence with 10-fold higher affinity than the monomeric PAX5 DBD when it is in a long DNA molecule. We suggest that the increased affinity results from interactions of one or more of the additional DBDs with neighboring non-specific sites in a long DNA molecule, and that this can account for the increased stability of PAX5-C20S chromatin binding compared with wild-type PAX5, resulting in DN activity by competition for binding to PAX5-target sites. Consistent with this model, the ALL-associated PAX5 fused to ETV6 or the multimerization domain of ETV6 SAM results in stable chromatin binding and DN activity. In addition, PAX5 DBD fused to artificial dimerization, trimerization and tetramerization domains results in parallel increases in the stability of chromatin binding and DN activity. Our studies suggest that oncogenic fusion proteins that retain the DBD of the transcription factor (TF) and the multimerization sequence of the partner protein can act in a DN manner by multimerizing and binding avidly to gene targets, preventing the normal TF from binding and inducing expression of its target genes. Inhibition of this multimeriztion may provide a novel therapeutic approach for cancers with this or similar fusion proteins.

Thyroid carcinoma cells often do not express thyroid-specific genes including sodium iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (TG), and thyrotropin-stimulating hormone receptor (TSHR). Treatment of thyroid carcinoma cells (four papillary and two anaplastic cell lines) with histone deacetylase inhibitors (SAHA or VPA) modestly induced the expression of the NIS gene. The promoter regions of the thyroid-specific genes contained binding sites for hepatocyte nuclear factor 3 beta (HNF3 beta)/forkhead box A2 (FoxA2), thyroid transcription factor 1 (TTF-1), and CCAAT/enhancer binding protein (C/EBP beta). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed decreased expression of HNF3 beta/FoxA2 and TTF-1 mRNA in papillary thyroid carcinoma cell lines, when compared with normal thyroid cells. Forced expression of these genes in papillary thyroid carcinoma cells inhibited their growth. Furthermore, the CpG island in the promoter region of HNF3 beta/FoxA2 was aberrantly methylated; and treatment with 5-aza-2-deoxycytidine (5-Az) induced its expression. Immunohistochemical staining showed that C/EBP beta was localised in the nucleus in normal thyroid cells but was detected in the cytoplasm in papillary thyroid carcinoma cells. Subcellular fractionation of papillary thyroid carcinoma cell lines also demonstrated high levels of expression of C/EBP beta in the cytoplasm, suggesting that a large proportion of C/EBP beta protein is inappropriately localised in the cytoplasm. In summary, these findings reveal novel abnormalities in thyroid carcinoma cells.

HIV-1 protease inhibitor (PI), nelfinavir (NFV) induced growth arrest and apoptosis of NCI-H460 and -H520, A549, EBC-1 and ABC-1 non-small-cell lung cancer (NSCLC) cells in association with upregulation of p21waf1, p27kip1 and p53, and downregulation of Bcl-2 and matrix metalloproteinase (MMP)-2 proteins. We found that NFV blocked Akt signalling in these cells as measured by Akt kinase assay with glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) as a substrate. To explore the role of Akt signalling in NFV-mediated growth inhibition of NSCLC cells, we blocked this signal pathway by transfection of Akt small interfering RNA (siRNA) in these cells; transient transfection of Akt siRNA in NCI-H460 cells decreased the level of Bcl-2 protein and slowed their proliferation compared to the nonspecific siRNA-transfected cells. Conversely, forced-expression of Akt partially reversed NFV-mediated growth inhibition of these cells, suggesting that Akt may be a molecular target of NFV in NSCLC cells. Also, we found that inhibition of Akt signalling by NFV enhanced the ability of docetaxel to inhibit the growth of NCI-H460 and -H520 cells, as measured by MTT assay. Importantly, NFV slowed the proliferation and induced apoptosis of NCI-H460 cells present as tumour xenografts in nude mice without adverse systemic effects. Taken together, this family of compounds might be useful for the treatment of individuals with NSCLC.

12-0-tetradecanoylphorbol-13-acetate (TPA) stimulates protein kinase C (PKC) which mediates apoptosis in androgen-sensitive LNCaP human prostate cancer cells. The downstream signals of PKC that mediate TPA-induced apoptosis in LNCaP cells are unclear. In this study, we found that TPA activates the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 pathway. To explore the possible role that the JNK/c-Jun/AP-1 signal pathway has on TPA-induced apoptosis in LNCaP cells, we stably transfected the scaffold protein, JNK interacting protein 1 (JIP-1), which binds to JNK inhibiting its ability to phosphorylate c-Jun. TPA (10(-9)-10(-7) mol l(-1)) caused phosphorylation of JNK in both wild-type and JIP-1-transfected (LNCaP-JIP-1) cells. It resulted in phosphorylation and upregulation of expression of c-Jun protein in the wild-type LNCaP cells, but not in the JIP-1-transfected LNCaP cells. In addition, upregulation of AP-1 reporter activity by TPA (10(-9) mol l(-1)) occurred in LNCaP cells but was abrogated in LNCaP-JIP-1 cells. Thus, TPA stimulated c-Jun through JNK, and JIP-1 effectively blocked JNK. TPA (10(-12)-10(-8) mol l(-1)) treatment of LNCaP cells caused their growth inhibition, cell cycle arrest, upregulation of p53 and p21waf1, and induction of apoptosis. All of these effects were significantly attenuated when LNCaP-JIP-1 cells were similarly treated with TPA. A previous study showed that c-Jun/AP-1 blocked androgen receptor (AR) signaling by inhibiting AR binding to AR response elements (AREs) of target genes including prostate-specific antigen (PSA). Therefore, we hypothesised that TPA would not be able to disrupt the AR signal pathway in LNCaP-JIP-1 cells. Contrary to expectation, TPA (10(-9)-10(-8) mol l(-1)) inhibited DHT-induced AREs reporter activity and decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, TPA, probably by stimulation of PKC, phosphorylates JNK, which phosphorylates and increases expression of c-Jun leading to AP-1 activity. Growth control of prostate cancer cells can be mediated through the JNK/c-Jun pathway, but androgen responsiveness of these cells can be independent of this pathway, suggesting that androgen independence in progressive prostate cancer may not occur through activation of this pathway.

The organic arsenical known as melarsoprol (Mel-B) is used to treat African trypanosomiasis. Recently, another arsenical, As2O3 was shown to be effective in treatment of acute promyelocytic leukaemia. We have investigated the anti-tumour activities of Mel-B either with or without all-trans-retinoic acid (ATRA) using the MCF-7 human breast cancer cells, as well as the PC-3 and DU 145 human prostate cancer cells both in vitro and in vivo. The antiproliferative effects of Mel-B and/or ATRA against breast and prostate cancer were tested in vitro using clonogenic assays and in vivo in triple immunodeficient mice. Furthermore, the mechanism of action of these compounds was studied by examining the cell cycle, levels of bcl-2, apoptosis and antiproliferative potency using a pulse-exposure assay. Clonogenic assays showed that the cancer cell lines were sensitive to the inhibitory effect of Mel-B (effective dose that inhibited 50% clonal growth [ED50]: 7 x 10(-9) M for MCF-7, 2 x 10(-7) M for PC-3, 3 x 10(-7) M for DU145 cells. Remarkably, the combination of Mel-B and ATRA had an enhanced antiproliferative activity against all three cancer cell lines. Furthermore, the combination of Mel-B and ATRA induced a high level of apoptosis in all three cell lines. Treatment of PC-3 and MCF-7 tumours growing in triple immunodeficient mice with Mel-B and ATRA either alone or in combination markedly retarded tumour size and weight of the tumours without major side-effects. In conclusion, our results suggest that either Mel-B alone or with ATRA may be a useful, novel therapy for breast and prostate cancers.

The secosteroid hormones, all-trans- and 9-cis-retinoic acid and vitamin D3, have demonstrated significant capacity to control proliferation in vitro of many solid tumour cell lines. Cooperative synergistic effects by these two ligands have been reported, and it is, therefore, possible that greater therapeutic effects could be achieved if these compounds were administered together. The role of retinoid-dependent anti-activator protein 1 (anti-AP-1) effects in controlling cancer cell proliferation appears significant. We have utilized an anti-AP-1 retinoid [2-(4,4-dimethyl-3,4-dihydro-2H-1 benzopyran-6-yl)carbonyl-2-(4-carboxyphenyl)-1,3,-dithiane; SR11238], which does not transactivate through a retinoic acid response element (RARE), and a potent vitamin D3 analogue [1alpha,25(OH)2-16-ene-23-yne-26,27-F6-19-nor-D3, code name LH] together at low, physiologically safer doses against a panel of prostate cancer cell lines that represent progressively more transformed phenotypes. The LNCaP (least transformed) and PC-3 (intermediately transformed) cell lines were synergistically inhibited in their clonal growth by the combination of LH and SR11238, whereas SR11238 alone was essentially inactive. DU-145 cells (most transformed) were completely insensitive to these analogues. LNCaP cells, but neither PC-3 nor DU-145, underwent apoptosis in the presence of LH and SR11238. Transactivation of the human osteocalcin vitamin D response element (VDRE) by LH was not enhanced in the presence of SR11238, although the expression of E-cadherin in these cells was additively up-regulated in the presence of both compounds. These data suggest the anti-AP-1 retinoid and the vitamin D3 analogue may naturally act synergistically to control cell proliferation, a process that is interrupted during transformation, and that this combination may form the basis for treatment of some androgen-independent prostate cancer.

Hormonally insensitive prostate cancer is a relatively slow-growing, but usually fatal, disease with no long-term treatment options. Transformation of normal prostate cells to a malignant phenotype often involves corruption of the apoptotic machineries. Bcl-2 protein is one of the key inhibitors of apoptosis and is often unregulated in advanced prostate cancer. The prostate cancer cell line DU-145 was used as a model of a hormonally insensitive, advanced prostate cancer. Cell growth in liquid culture was significantly inhibited by antisense Bcl-2 oligonucleotides compared with control sense oligonucleotides; inhibition by these oligonucleotides was significantly enhanced on combination with the synthetic retinoid N-(2-hydroxyphenyl)all-trans-retinamide (2-HPR). Interestingly, growth inhibition occurred in the absence of apoptosis as measured using two assay techniques. We hypothesize that in these recalcitrant cells the apoptotic pathway is compromised at several levels, and Bcl-2 may play another role in promoting cell growth. The use of Bcl-2 antisense oligonucleotides plus 2-HPR may provide a novel approach to therapy of hormone-resistant prostate cancer.

Identification of the key genetic alterations leading to ovarian cancer is in its infancy. Polymerase chain reaction (PCR)-based analysis of loss of heterozygosity (LOH) is a powerful method for detecting regions of altered tumour-suppressor genes. Focusing on chromosome 12, we examined 23 ovarian cancer samples for LOH using 31 highly polymorphic microsatellite markers and found the chromosomal localization of two putative tumour-suppressor genes. Two commonly deleted regions were 12p12.3-13.1 in 6/23 (26%) and 12q23-ter in 7/23 (30%) samples. LOH on chromosome 12 was more common in late-stage ovarian carcinomas. The region of LOH at 12p12.3-13.1 includes the genes that code for the ETS-family transcriptional factor, known as TEL, and the cyclin-dependent kinase inhibitor, known as p27Kip1. Mutational analysis of both TEL and p27Kip1 using single-strand conformation polymorphism (SSCP) showed no abnormalities, suggesting that the altered gene in this region is neither of these genes. Taken together, our data suggest that new tumour-suppressor genes in the region of chromosomes 12p12.3-13.1 and 12q23-ter may be involved in the development of ovarian cancer.

The p53 is a nuclear protein that is associated with normal cellular proliferation and can cooperate with Ha-ras in causing cellular transformation in vitro. Lineage association is known to exist between p53 expression and normal lymphopoiesis, but not myelopoiesis. We studied the expression of p53 using chronic myelogenous leukemia (CML) cell lines, somatic hybrids of these cells, and leukemic cells from CML patients. Lymphoid CML lines expressed both p53 mRNA and protein. We also analyzed p53 synthesis by two B-lymphoid lines from the same CML patient; cells of one line were derived from the neoplastic clone, cells of the other were derived from the normal clone. Both synthesized equal amounts of a phosphorylated p53 protein. None of the myeloid CML lines expressed detectable p53 protein and two of four expressed negligible p53 mRNA. Two other myeloid CML lines and myeloid cells from three of four patients expressed p53 mRNA. These findings suggest that expression of the gene is not regulated normally in CML. Several approaches were pursued to explore the differential expression of p53. Southern blot analyses showed no gross alterations in the p53 gene from cells of either the expressing or the nonexpressing lines. No difference in the pattern of demethylated CpG sites was noted in the region of the p53 gene in cells from K562 (myeloid p53 nonexpressor) and in BV173 (lymphoid p53 expressor). The sites of demethylation clustered in and around the p53 promoter in both cell lines. Somatic hybrids formed between a p53 mRNA nonexpressor myeloid line (K562) and the parental p53 expressor lymphoid lines (Daudi, PUT) produced p53 mRNA and protein, suggesting that p53 is a dominantly expressed protein and that lack of expression in myeloid cells is not mediated by a trans-acting negative regulatory protein. DNA transfection experiments performed using the indicator gene chloramphenicol acetyltransferase attached to promoter sequences of p53 showed that these constructs were equally activated in BV173 (p53 expressor) and K562 (p53 mRNA nonexpressor). The mechanism of p53 regulation in CML remains unclear.

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 5.7 X 10(-9) M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number. The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.