Histone deacetylases and cancer: causes and therapies.

Together, histone acetyltransferases and histone deacetylases (HDACs) determine the acetylation status of histones. This acetylation affects the regulation of gene expression, and inhibitors of HDACs have been found to cause growth arrest, differentiation and/or apoptosis of many tumours cells by altering the transcription of a small number of genes. HDAC inhibitors are proving to be an exciting therapeutic approach to cancer, but how do they exert this effect?

Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations, invariably involving the retinoic acid receptor alpha (RAR alpha) gene fused to one of several distinct loci, including the PML or PLZF genes, involved in t(15;17) or t(11;17), respectively. Patients with t(15;17) APL respond well to retinoic acid (RA) and other treatments, whereas those with t(11;17) APL do not. The PML-RAR alpha and PLZF-RAR alpha fusion oncoproteins function as aberrant transcriptional repressors, in part by recruiting nuclear receptor-transcriptional corepressors and histone deacetylases (HDACs). Transgenic mice harboring the RAR alpha fusion genes develop forms of leukemia that faithfully recapitulate both the clinical features and the response to RA observed in humans with the corresponding translocations. Here, we investigated the effects of HDAC inhibitors (HDACIs) in vitro and in these animal models. In cells from PLZF-RAR alpha/RAR alpha-PLZF transgenic mice and cells harboring t(15;17), HDACIs induced apoptosis and dramatic growth inhibition, effects that could be potentiated by RA. HDACIs also increased RA-induced differentiation. HDACIs, but not RA, induced accumulation of acetylated histones. Using microarray analysis, we identified genes induced by RA, HDACIs, or both together. In combination with RA, all HDACIs tested overcame the transcriptional repression exerted by the RAR alpha fusion oncoproteins. In vivo, HDACIs induced accumulation of acetylated histones in target organs. Strikingly, this combination of agents induced leukemia remission and prolonged survival, without apparent toxic side effects.

Suppression of cyclin-dependent kinase 4 during induced differentiation of erythroleukemia cells.

Differentiation of murine erythroleukemia cells induced by hexamethylene bisacetamide (HMBA) is associated with accumulation of underphosphorylated retinoblastoma protein (pRB) and an increase in retinoblastoma (RB) gene expression. Here we show that HMBA causes a rapid decrease in the level of cyclin-dependent kinase 4 (cdk4) protein. This decrease results from decreased stability of the protein, while the rate of synthesis of the protein is not affected by HMBA. The decrease in the level of cdk4 protein is followed by suppression of the pRB kinase activity associated with cdk4. Cyclin D3, which can bind and activated cdk4, is increased in HMBA-induced cells and is found in complex with pRB and the transcription factor E2F. In uninduced cells cyclin D3 complexes with pRB and E2F are barely detected. At the later stages of differentiation, MEL cells become arrested in G1 and cdk2 kinase activity is suppressed; this is accompanied by a decrease in the level of cyclin A and cdk2 proteins. Cells transfected with cdk4, which continue to overexpress cdk4 protein during culture with HMBA, are resistant to HMBA-induced differentiation. In contrast, overexpression of cdk2 protein does not inhibit induced differentiation. These findings suggest that suppression of cdk4 is a critical event in the pathway leading to terminal differentiation of erythroleukemia cells.

Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells.

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.

Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells.

Histone deacetylase (HDAC) inhibitors have been shown to be potent inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells in vitro and in vivo. One class of HDAC inhibitors, hydroxamic acid-based hybrid polar compounds (HPCs), induce differentiation at micromolar or lower concentrations. Studies (x-ray crystallographic) showed that the catalytic site of HDAC has a tubular structure with a zinc atom at its base and that these HDAC inhibitors, such as suberoylanilide hydroxamic acid and trichostatin A, fit into this structure with the hydroxamic moiety of the inhibitor binding to the zinc. HDAC inhibitors cause acetylated histones to accumulate in both tumor and normal tissues, and this accumulation can be used as a marker of the biologic activity of the HDAC inhibitors. Hydroxamic acid-based HPCs act selectively to inhibit tumor cell growth at levels that have little or no toxicity for normal cells. These compounds also act selectively on gene expression, altering the expression of only about 2% of the genes expressed in cultured tumor cells. In general, chromatin fractions enriched in actively transcribed genes are also enriched in highly acetylated core histones, whereas silent genes are associated with nucleosomes with a low level of acetylation. However, HDACs can also acetylate proteins other than histones in nucleosomes. The role that these other targets play in the induction of cell growth arrest, differentiation, and/or apoptotic cell death has not been determined. Our working hypothesis is that inhibition of HDAC activity leads to the modulation of expression of a specific set of genes that, in turn, result in growth arrest, differentiation, and/or apoptotic cell death. The hydroxamic acid-based HPCs are potentially effective agents for cancer therapy and, possibly, cancer chemoprevention.

Prognostic significance of transcription factor E2F-1 in bladder cancer: genotypic and phenotypic characterization.

BACKGROUND: We sought to identify and characterize potential alterations in E2F-1, a transcription factor that binds to the retinoblastoma protein (pRB), in bladder neoplasms and to elucidate a possible role for E2F-1 as an oncogene or a tumor suppressor gene. METHODS: Tumor samples from 133 evaluable patients with bladder cancer were analyzed for E2F-1 gene mutations by use of polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, tumors were studied for E2F-1 and pRB protein expression by use of immunohistochemistry. Results from the above analyses were correlated with clinicopathologic parameters and outcome. All P values are two-sided. RESULTS: A polymorphism, consisting of a nucleotide change at amino acid codon 393 in exon 7 (GGC-->AGC [Gly-->Ser]), was identified in seven of 133 case patients, being present in both tumor and corresponding normal tissues. No bandshifts were identified in the nuclear-localization or DNA-binding domains on PCR-SSCP analysis. On immunohistochemical analysis, E2F-1 nuclear reactivity was observed in less than 5% of the cells from 53 tumors and in 5%-75% of the cells from the remaining 80 tumors. The pattern of E2F-1 protein expression was not altered in relation to the identified polymorphism. pRB nuclear reactivity greater than 20% (of tumor cells stained) was present in 66% of the samples. E2F-1 nuclear reactivity correlated inversely with the percentage of cells showing pRB reactivity (Kendall tau(b) = -0.18; P = .019). On multivariate analysis, patients with lower E2F-1 reactivity had statistically significantly increased risks of progression to metastases (P = .001) and death (P = .02). CONCLUSIONS: E2F-1 alterations occur at the phenotypic level, rather than at the genotypic level, in bladder cancer. The adverse outcome for patients whose tumors exhibit low E2F-1 nuclear expression suggests a possible tumor suppressor role for E2F-1 in bladder cancer.

Multiple mechanisms of resistance to cis-diamminedichloroplatinum(II) in murine leukemia L1210 cells.

As an experimental model for resistance to cis-diamminedichloroplatinum(II) (cis-DDP), murine leukemia L1210 cells have been exposed to a stepwise increase in cis-DDP concentration to produce a variety of resistant cell lines. Intraspecies hybrids of the sensitive and resistant cells were made to determine whether cis-DDP resistance is a dominant or recessive trait. Hybrid cells displayed a partial degree of resistance as compared to the parental cells. To determine whether this was due to a single codominant trait or contribution from a variety of resistance mechanisms, the cells and hybrids were investigated for alterations in the accumulation of drug, as well as alterations in glutathione levels which might inactivate the drug. The cis-DDP-resistant cells demonstrated both a 50% reduction in accumulation of drug and a 1.7-fold increase in intracellular glutathione. Reducing the glutathione levels in these cells with buthionine sulfoximine did not sensitize them to cis-DDP. The hybrid cells had the same accumulation and the same levels of glutathione as the cis-DDP-sensitive cells. Parallel studies were performed with cells resistant to 1,2-diaminocyclohexaneplatinum(II) analogues. These cells also demonstrated reduced drug accumulation but no increase in glutathione. Therefore, both a decrease in accumulation and increase in glutathione may mediate resistance. Both mechanisms represent recessive traits as demonstrated in the cell hybrids. These mechanisms can only account for a small part of the resistance in these cells. A major, dominant mechanism occurs after the DNA has been platinated, but it remains to be determined whether this involves DNA repair, postreplication repair, or some other as yet unidentified process.

Two cytodifferentiation agent-induced pathways, differentiation and apoptosis, are distinguished by the expression of human papillomavirus 16 E7 in human bladder carcinoma cells.

Many transformed cells have been found to lose the capacity to proliferate and undergo differentiation following exposure to hybrid polar agents. This study investigates the mechanism by which hexamethylene bisacetamide (HMBA) suppresses the proliferation of the human bladder carcinoma line, T24. We found that following a 24-h exposure to HMBA, T24 proliferation was inhibited, and cells arrested in G1 phase and underwent morphological maturation. HMBA-induced cessation of proliferation was mediated, in part, by effects on cell cycle regulatory proteins. In T24 cells cultured without HMBA, E2F complexes predominantly with p107. In culture with inducer, p107 protein decreased, pRB and p130 were converted to underphosphorylated forms, and E2F was shifted into complexes with pRB and p130. To determine whether the formation of pRB:E2F and p130:E2F complexes was required for the HMBA-induced G1 arrest, the ability of the pocket proteins to bind E2F was blocked by enforced expression of human papillomavirus 16 E7. Following culture with HMBA, the T24 clones expressing E7 died, whereas vector-alone T24 clones arrested in G1 phase. T24/E7-1 cells did not form pRB:E2F or p130:E2F complexes upon culture with HMBA; rather, E2F was present in its free form. T24/E7-1 cells cultured with HMBA initially accumulate in G1. By day 2, they have entered into S phase, and by day 3, over 80% of the cells became apoptotic. Taken together, these studies enlarge the repertoire of demonstrated developmental pathways that may be triggered in transformed cells, depending upon their molecular status, and may provide potential therapeutic opportunities for cancer.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces differentiation of human breast cancer cells.

Histone deacetylase (HDACs) regulate histone acetylation by catalyzing the removal of acetyl groups on the NH(2)-terminal lysine residues of the core nucleosomal histones. Modulation of the acetylation status of core histones is involved in the regulation of the transcriptional activity of certain genes. HDAC activity is generally associated with transcriptional repression. Aberrant recruitment of HDAC activity has been associated with the development of certain human cancers. We have developed a class of HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA), that were initially identified based on their ability to induce differentiation of cultured murine erythroleukemia cells. Additional studies have demonstrated that SAHA inhibits the growth of tumors in rodents. In this study we have examined the effects of SAHA on MCF-7 human breast cancer cells. We found that SAHA causes the inhibition of proliferation, accumulation of cells in a dose-dependent manner in G(1) then G(2)-M phase of the cell cycle, and induction of milk fat globule protein, milk fat membrane globule protein, and lipid droplets. Growth inhibition was associated with morphological changes including the flattening and enlargement of the cytoplasm, and a decrease in the nuclear:cytoplasmic ratio. Withdrawal of SAHA led to reentry of cells into the cell cycle and reversal to a less differentiated phenotype. SAHA induced differentiation in the estrogen receptor-negative cell line SKBr-3 and the retinoblastoma-negative cell line MDA-468. We propose that SAHA has profound antiproliferative activity by causing these cells to undergo cell cycle arrest and differentiation that is dependent on the presence of SAHA. SAHA and other HDAC inhibitors are currently in Phase I clinical trials. These findings may impact the clinical use of these drugs.

Differential modulation of protein kinase C isoforms in erythroleukemia during induced differentiation.

Induction of erythroid differentiation of murine erythroleukemia cells (MELC) by exposure to hexamethylene bisacetamide (HMBA) involves the modulation of protein kinase C (PKC) activity. Using immuno- and Northern blot techniques, we have demonstrated that MELC express a pattern of PKC isoforms which includes PKC alpha, PKC delta, PKC epsilon, PKC zeta, and PKC eta. We show that MELC resistant to induction by HMBA express significantly less of the nPKC isoform, PKC delta, and slightly less PKC epsilon. Recovery of HMBA sensitivity is associated with reexpression of PKC delta protein. Upon exposure to HMBA, there is a fall in cytosolic PKC delta and PKC epsilon accompanied by a transient increase in membrane-associated forms of these PKC isoforms. HMBA-resistant MELC fail to display this isoform-specific translocation of PKC. Induction of differentiation is accompanied, over the next 24 h of exposure to HMBA, by a progressive fall in cellular PKC activity, associated with a progressive fall in the cellular content of PKC delta, PKC epsilon, and PKC zeta. These studies suggest that PKC delta, and possibly PKC epsilon and PKC zeta as well, play a role in the pathway of HMBA-mediated terminal cell differentiation of MELC.

The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma xenografts in vivo, alone and synergistically with all-trans retinoic acid.

Histone deacetylase inhibitors (HDACIs) inhibit the growth of a variety of transformed cells in culture. We demonstrated previously that the hybrid-polar HDACI m-carboxycinnamic acid bis-hydroxamide (CBHA) induces apoptosis of human neuroblastoma in vitro and is effective in lower doses when combined with retinoids. The current study investigates the effect of CBHA on the growth of human neuroblastoma in vivo, both alone and in combination with all-trans retinoic acid (atRA), using a severe combined immunodeficiency-mouse xenograft model. CBHA (50, 100, and 200 mg/kg/day) inhibited growth of SMS-KCN-69n tumor xenografts in a dose-dependent fashion, with 200 mg/kg CBHA resulting in a complete suppression of tumor growth. The efficacy of 50 and 100 mg/kg CBHA was enhanced by the addition of 2.5 mg/kg atRA. This dose of atRA was ineffective when administered alone. Treatment was accompanied by mild weight loss in all groups except the lowest dose of CBHA. Our results suggest HDACIs alone or combined with retinoids may have therapeutic utility for neuroblastoma.

Hybrid polar histone deacetylase inhibitor induces apoptosis and CD95/CD95 ligand expression in human neuroblastoma.

Inhibitors of histone deacetylase (HDAC) have been shown to have both apoptotic and differentiating effects on various tumor cells. M-carboxycinnamic acid bishydroxamide (CBHA) is a recently developed hybrid polar compound structurally related to hexamethylene bisacetamide. CBHA is a potent inhibitor of HDAC activity. CBHA induces cellular growth arrest and differentiation in model tumor systems. We undertook an investigation of the effects of CBHA on human neuroblastoma cell lines in vitro. When added to cultures of a panel of neuroblastoma cell lines, CBHA induced the accumulation of acetylated histones H3 and H4, consistent with the inhibition of HDAC. Concentrations of CBHA between 0.5 microM and 4 microM led to apoptosis in nine of nine neuroblastoma cell lines. Apoptosis was assessed by DNA fragmentation analysis and the appearance of a sub-G1 (<2N ploidy) population by flow cytometric analysis. The addition of a caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) completely abrogated CBHA-induced apoptosis in three of three cell lines. The addition of cycloheximide greatly reduced CBHA-induced apoptosis, suggesting that apoptotic induction was dependent on de novo protein synthesis. In addition, CBHA induced the expression of both CD95 (APO-1/Fas) and CD95 ligand within 12 h. The effect of CBHA on human neuroblastoma cells suggests that this agent and structurally related synthetic hybrid polar compounds have therapeutic potential for the treatment of this malignancy.

Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo.

Suberoylanilide hydroxamic acid (SAHA) is the prototype of a family of hybrid polar compounds that induce growth arrest in transformed cells and show promise for the treatment of cancer. SAHA induces differentiation and/or apoptosis in certain transformed cells in culture and is a potent inhibitor of histone deacetylases. In this study, we examined the effects of SAHA on the growth of human prostate cancer cells in culture and on the growth of the CWR22 human prostate xenograft in nude mice. SAHA suppressed the growth of the LNCaP, PC-3, and TSU-Pr1 cell lines at micromolar concentrations (2.5-7.5 microM). SAHA induced dose-dependent cell death in the LNCaP cells. In mice with transplanted CWR222 human prostate tumors, SAHA (25, 50, and 100 mg/kg/day) caused significant suppression of tumor growth compared with mice receiving vehicle alone; treatment with 50 mg/kg/day resulted in a 97% reduction in the mean final tumor volume compared with controls. At this dose, there was no detectable toxicity as evaluated by weight gain and necropsy examination. Increased accumulation of acetylated core histones was detected in the CWR22 tumors within 6 h of SAHA administration. SAHA induced prostate-specific antigen mRNA expression in CWR22 prostate cancer cells, resulting in higher levels of serum prostate-specific antigen than predicted from tumor volume alone. The results suggest that hydroxamic acid-based hybrid polar compounds inhibit prostate cancer cell growth and may be useful, relatively nontoxic agents for the treatment of prostate carcinoma.

Modulation of the c-myb, c-myc and p53 mRNA and protein levels during induced murine erythroleukemia cell differentiation.

The induction of murine erythroleukemia cells (MELC) to terminal differentiation by hexamethylene bisacetamide (HMBA) is accompanied by changes in the levels of c-myb and c-myc mRNA, and in p53 protein levels. We simultaneously examined the effects of HMBA on modulation of c-myb, c-myc and p53 mRNA and protein levels, and examined the relationship between these changes and commitment to terminal cell division. In MELC cultured with HMBA, c-myb protein levels paralleled c-myb mRNA levels except at 24h, when the protein level was equivalent to the level in control cultures, whereas the mRNA had decreased. The c-myc protein paralleled c-myc mRNA throughout induction. The p53 mRNA and protein behaved in a discordant fashion. The p53 protein decreased to very low levels between 4 and 8 h and remained low, while the mRNA, which initially decreased, reaccumulated by 24 and 48 h. Transfer of MELC after 12 to 48 h of culture with HMBA to medium without inducer resulted in rapid (less than 3 h) reaccumulation of the c-myb mRNA, c-myb protein, and p53 protein, and cessation of recruitment of cells to commitment. Cells already induced to commit to terminal differentiation continued to express the differentiated phenotype.

Induction of apoptosis in U937 human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and p21CIP1, but independent of p53.

Determinants of differentiation and apoptosis in myelomonocytic leukemia cells (U937) exposed to the novel hybrid polar compound SAHA (suberoylanilide hydroxamic acid) have been examined. In contrast to hexamethylenbisacetamide (HMBA), SAHA-related maturation was limited and accompanied by marked cytoxicity. SAHA-mediated apoptosis occurred within the G0G1 and S phase populations, and was associated with decreased mitochondrial membrane potential, caspase-3 activation, PARP degradation, hypophosphorylation/cleavage of pRB, and down-regulation of c-Myc, c-Myb, and B-Myb. Enforced expression of Bcl-2 or Bcl-XL inhibited SAHA-induced apoptosis, but only modestly potentiated differentiation. While SAHA induced the cyclin-dependent kinase inhibitor p21CIP1, antisense ablation of this CDKI increased, rather than decreased, SAHA-related lethality. In contrast, conditional expression of wild-type p53 failed to modify SAHA actions, but markedly potentiated HMBA-induced apoptosis. Finally, SAHA modestly increased expression/activation of the stress-activated protein kinase (SAPK/JNK); moreover, SAHA-related lethality was partially attenuated by a dominant-negative c-Jun mutant protein (TAM67). SAHA did not stimulate mitogen-activated protein kinase (MAPK), nor was lethality diminished by the specific MEK/MAPK inhibitor PD98059. These findings indicate that SAHA potently induces apoptosis in human leukemia cells via a pathway that is p53-independent but at least partially regulated by Bcl-2/Bcl-XL, p21CIP1, and the c-Jun/AP-1 signaling cascade.

Induced differentiation, the cell cycle, and the treatment of cancer.

Hybrid polar compounds, of which hexamethylene bisacetamide (HMBA) is the prototype, have been shown to be potent inducers of differentiation of many types of transformed cells. With virus-transformed murine erythroleukemia cells as a model, HMBA was shown to cause these cells to arrest in G1 phase and express globin genes. HMBA action involves modulation of factors regulating G1 to S phase progression, including a decrease in the G1 cyclin-dependent kinase 4 accumulation of underphosphorylated retinoblastoma protein, and an increase in the level of both retinoblastoma protein and the related protein, p107. In turn, p107 complexes with transcription factors such as E2F and, presumably, inhibits transcriptional activity of these factors for genes whose products are required for DNA synthesis. This provides a possible mechanism for HMBA-induced terminal cell division of transformed cells. Evidence that hybrid polar compounds have therapeutic potential for cancer treatment is also reviewed.

Inhibition of transformed cell growth and induction of cellular differentiation by pyroxamide, an inhibitor of histone deacetylase.

PURPOSE: We have synthesized a series of hybrid polar compounds that induce differentiation and/or apoptosis of various transformed cells. These agents are also potent inhibitors of histone deacetylases (HDACs). Pyroxamide (suberoyl-3-aminopyridineamide hydroxamic acid) is a new member of this class of compounds that is currently under development as an anticancer agent. We investigated the activity of pyroxamide as an inducer of differentiation and/or apoptosis in transformed cells. EXPERIMENTAL DESIGN AND RESULTS: Pyroxamide, at micromolar concentrations, induced terminal differentiation in murine erythroleukemia (MEL) cells and caused growth inhibition by cell cycle arrest and/or apoptosis in MEL, prostate carcinoma, bladder carcinoma, and neuroblastoma cells. Administration of pyroxamide (100 or 200 mg/kg/day) to nude mice at doses that caused little evident toxicity significantly suppressed the growth of s.c. CWR22 prostate cancer xenografts. Despite the potent growth-inhibitory effects of pyroxamide in this tumor model, serum prostate-specific antigen levels in control versus pyroxamide-treated mice were not significantly different. Pyroxamide is a potent inhibitor of affinity-purified HDAC1 (ID(50) = 100 nM) and causes the accumulation of acetylated core histones in MEL cells cultured with the agent. Human CWR22 prostate tumor xenografts from mice treated with pyroxamide (100 or 200 mg/kg/day) showed increased levels of histone acetylation and increased expression of the cell cycle regulator p21/WAF1, compared with tumors from vehicle-treated control animals. CONCLUSIONS: The findings suggest that pyroxamide may be a useful agent for the treatment of malignancy and that induction of p21/WAF1 in transformed cells by pyroxamide may contribute to the antitumor effects of this agent.

Cloning of the cDNA encoding phenylalanyl tRNA synthetase regulatory alpha-subunit-like protein whose expression is down-regulated during differentiation.

Hybrid polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA), induce differentiation of transformed cells. Differential display of RNA was used to identify genes whose expression is changed during SAHA-induced differentiation of murine erythroleukemia (MEL) cells. One such cDNA was identified whose mRNA level decreased by 50% after 8h of SAHA treatment as determined by Northern blot analysis. The full-length cDNA (1944bp in length) was cloned by sequencing of an EST clone and rapid amplification of 5' cDNA ends (5'-RACE). The predicted amino acid sequence is 589 amino acids and shares 45% identity with the yeast cytoplasmic phenylalanyl tRNA synthetase (PheRS) regulatory alpha-subunit. Human EST clones which share over 90% identity of predicted amino acid sequence with this cDNA map to chromosome 2 near the paired box homeotic gene 3 (PAX3) locus, a region syngenic to mouse chromosome 1. This is the first report of the cloning of the full-length cDNA for the murine PheRS regulatory alpha-subunit-like protein. The level of PheRS alpha-subunit-like mRNA is regulated during differentiation but not during cell cycle progression.

Cell cycle regulatory proteins are targets for induced differentiation of transformed cells: Molecular and clinical studies employing hybrid polar compounds.

Considerable progress has been made toward elucidating the pathway of induction of terminal differentiation of transformed cells by hybrid polar compounds such as hexamethylene bisacetamide (HMBA). HMBA alters factors controlling G1-to-S phase transition, leading to G1 arrest and inhibition of DNA synthesis. Among the inducer-mediated changes, suppression of cyclin-dependent kinase cdk4, which may be required for phosphorylation of the retinoblastoma protein pRB and perhaps p107, is critical in the pathway of terminal differentiation. HMBA induces an increase in the level of p21 which inhibits cyclin-dependent kinase activity and, in turn, may cause cells to arrest in G1. p107 complexes with transcription factor E2F, which may alter E2F-dependent gene transcription. the relationship of the inducer-mediated changes in cyclins, cdks, cyclin-cdk inhibitors and transcription factors to the expression of differentiation-specific genes has not yet been established. The hybrid polar compounds are potent inducers of differentiation of a wide variety of transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients. A second generation of hybrid polar compounds have been synthesized which are up to 1000 fold more potent than HMBA on a molar basis as inducers of murine erythroleukemia (MEL) cells and other transformed cells in vitro. The potential of these compounds as clinically useful inducers of differentiation of cancer cells is under study.

Chemoprevention of carcinogen-induced mammary tumorigenesis by the hybrid polar cytodifferentiation agent, suberanilohydroxamic acid (SAHA).

Hybrid Polar Cytodifferentiation (HPC) agents represent a novel class of anticancer compounds which act by inducing terminal differentiation and/or apoptosis rather than by cytotoxic action. Among these are HPC agents such as hexamethylenebisacetamide (HMBA) and more potent 2nd generation hybrid/polar compounds such as suberanilohydroxamic acid (SAHA). As of the present, most studies on HPC agents have focused on cancers of the hematopoietic system rather than solid epithelial tumors. The objective of the present study therefore was to assess the chemopreventive action of these two related compounds in the N-methylnitrosourea (NMU)-induced rat mammary tumor model. Female Sprague-Dawley rats were fed diets containing 450 and 900 ppm, SAHA and 1000 and 2000 ppm HMBA, starting one week prior to NMU administration and continued for a period of 18 weeks. Mammary tumor development was monitored by palpation throughout the study, and at termination tumor incidence, number, multiplicity, latency and volume were determined. Weight gain was measured biweekly throughout the study. The salient results were as follows: SAHA at 900 ppm reduced NMU-induced mammary tumor incidence by 40%, total tumors by 66%, mean tumor multiplicity by 43% and mean tumor volume by 78%, with no detectable toxic side effects. HMBA exerted no tumor inhibiting effects at either concentration. This study represents the first demonstration that an HPC agent, namely SAHA, can inhibit the development of a chemically-induced, solid, epithelial tumor, at a relatively low dose (approximately 13 mgs/rat/day) without untoward side effects.