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.

Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids.

The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.

Comparing patients' experience of mental health services in England: a five-Trust survey.

The implementation of the Care Programme Approach (CPA) in English mental health services has been slow to proceed despite general support, both in England and in other countries, of its principles of good practice. This study set out to evaluate the implementation of the CPA directly from patients' experience using the "Your Treatment and Care" assessment tool. The results of a survey of 503 patients across five NHS Trusts in England showed that many patients did not have a copy of their care plan and had not been involved in the care planning procedure. Many reported shortcomings in their experience of their key worker and their psychiatrist. However, there was substantial variation in experience across services. "Your Treatment and Care" showed good internal reliability, was acceptable to users, and appeared to be able to access actual experiences better than a traditional "satisfaction" item. It appears to be very useful as a benchmarking tool and is now being used in services across the UK, the USA and Australia.

A simple method of evaluating patients' perceptions of their treatment and care.

Surveys of patient satisfaction have become commonplace in mental health services. However, questions about the appropriateness of "satisfaction" as a concept and its use as an approach to evaluating the quality of service provision remain as pertinent as ever. Although surveys of patients of mental health services routinely generate high levels of apparent satisfaction, noncompliance with treatment and medication continues to be a major difficulty in psychiatric care. To evaluate services properly, there is a need to access patients' dissatisfaction to determine whether it is a more valid indicator of quality of services and a better predictor of noncompliance. However, a method that is not time consuming and costly is also required. The authors introduce a new patient evaluation tool, "Your Treatment and Care," which requires patients to report on their direct experience of care according to a set of principles of good practice. The tool forms part of a broader package for obtaining patients' views of their own problems, need for care, quality of care received, and health and social outcomes. The authors also report the results of a survey using this tool. Responses of 75 patients of a psychiatric hospital in the United States are presented and compared with results from a similar survey in the United Kingdom. The comparisons show striking differences in patients' perceptions of quality of treatment and care, both within each population and across clinical contexts.

Photoaffinity labeling and mass spectrometry identify ribosomal protein S3 as a potential target for hybrid polar cytodifferentiation agents.

The ability of a novel class of hybrid polar compounds (HPCs) to induce differentiation and consequent cessation of proliferation of transformed cells has led to their development as potential chemotherapeutic agents in the treatment of cancer. Suberoylanilide hydroxamic acid (SAHA) is a prototype of a family of hydroxamic acid based compounds (SAHA-like HPCs) that can, at micromolar concentrations, induce a variety of transformed cell lines to differentiate. The mechanism of action of the HPCs is not entirely understood. Searching for a cellular target of the SAHA-like HPCs, we synthesized a photoaffinity labeling reagent structurally based on SAHA, and probed for SAHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity labeling in cell free extracts identified a 32-kDa protein (p32) that was specifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction. p32 was partially purified and identified by mass spectrometry as the 40 S ribosomal protein S3. Expression of epitope-tagged S3 in bacterial lysates followed by photoaffinity labeling confirmed its specific labeling. Identification of a cytodifferentiation agent target may shed light on the mechanism by which the SAHA-like HPCs exert their antitumor effects.

A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases.

Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity in vitro. Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.

Second generation hybrid polar compounds are potent inducers of transformed cell differentiation.

Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL) cells and a wide variety of other transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients, but is not an adequate therapeutic agent because of dose-limiting toxicity. We report on a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2 microM, and 4 microM, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p2l protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of hemoglobin. However, based upon their effective concentrations, the cross-resistance or sensitivity of an HMBA-resistant MEL cell variant, and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in selecting and planning prospective preclinical and clinical studies toward the treatment of cancer by differentiation therapy.

A needs survey among patients in Leros asylum.

The recent exposure of the plight of inmates living in poor conditions at the state asylum on the Greek island of Leros has caused public and professional outrage. If Greece is to avoid mistakes made by other countries, the plans for rehabilitating the patients and closing the hospital should from the outset include identification of the precise needs of patients for support and care. The survey of the patients' characteristics and needs for care found that most patients had no outside friends or relatives, and most were unable to perform basic daily skills. They shared many basic characteristics, however, with a large sample of the long-stay population in the UK, and 25% were thought to be able to live independently.

Planning for community care: the Community Placement Questionnaire.

The article reviews methodological issues relating to planning surveys of long-stay populations and describes the development of an assessment instrument, the Community Placement Questionnaire, designed to aid planning for long-stay patients resident in hospitals scheduled for rundown or closure. Reliability and validity data are presented and the advantages and limitations of the instrument are discussed.

Planning for community care. Long-stay populations of hospitals scheduled for rundown or closure.

Using the Community Placement Questionnaire, the long-stay populations of five hospitals were surveyed. The results suggest that there is little need for large hospitals if adequate community provision is made. However, a small number of patients continue to accumulate for whom community placement is hard to envisage. Investigating the characteristics of the 'new long-stay' patients suggests that the usual definition should be extended to include those over 65 years old with no diagnosis of dementia and those in hospital for 1-10 years. About 20% of 'new long-stay' patients have organic diagnoses and the needs of this group require assessment.