Development and optimisation of a reception testing protocol designed to eliminate HCV in the UK prison population.

Background & Aims: Micro-elimination of hepatitis C virus (HCV) in high-risk populations is a feasible approach towards achieving the World Health Organization's targets for viral hepatitis elimination by 2030. Prisons represent an area of high HCV prevalence and so initiatives that improve testing and treatment of residents are needed to eliminate HCV from prisons. This initiative aimed to improve the HCV screening and treatment rates of new residents arriving at prisons in England. Methods: A rapid test and treat pathway was developed and implemented in 47 prisons in England between May 2019 and October 2021 as a healthcare service improvement initiative. Prison healthcare staff performed opt-out HCV testing for all new residents at each prison within 7 days of arrival, and those who were positive for HCV RNA were offered treatment with direct-acting antivirals (DAAs). The Hepatitis C Trust provided peer support for all residents on treatment and those who were released into the community. Results: Of 107,260 new arrivals, 98,882 (92.2%) were offered HCV antibody testing, 63,137 (63.9%) were tested and 1,848 were treated. Testing rates increased from 53.7% in Year 1 to 86.0% in Year 3. Between May 2020 and October 2021, 40,727 residents were tested, 2,286 residents were positive for HCV antibodies and 940 residents were HCV RNA positive, giving an antibody prevalence of 5.6% and an RNA prevalence of 2.3%. A total of 921 residents were referred for treatment and 915 initiated DAA treatment (97.3% of whom were HCV RNA positive). Conclusions: This initiative showed that an opt-out HCV test and treat initiative in prison receptions is feasible and can be adapted to the needs of individual prisons as a viable way to achieve HCV micro-elimination. Impact and implications: Prisons represent an area of high HCV prevalence and so initiatives that improve testing and treatment of residents are needed to eliminate HCV from prisons. The reception testing protocol improved HCV screening in new arrivals across 47 prisons in England and could be a viable way for countries to achieve HCV micro-elimination in their prison systems. The reception testing protocol presented here can be adapted to the individual needs of prisons, globally, to improve HCV screening and treatment in this setting.

How Referring Providers Choose Specialists for Their Patients: a Systematic Review.

BACKGROUND: Physician referrals are a critical step in directing patients to high-quality specialists. Despite efforts to encourage referrals to high-volume hospitals, many patients receive treatment at low-volume centers with worse outcomes. We aimed to determine the most important factors considered by referring providers when selecting specialists for their patients through a systematic review of medical and surgical literature. METHODS: PubMed and Embase were searched from January 2000 to July 2021 using terms related to referrals, specialty, surgery, primary care, and decision-making. We included survey and interview studies reporting the factors considered by healthcare providers as they refer patients to specialists in the USA. Studies were screened by two independent reviewers. Quality was assessed using the CASP Checklist. A qualitative thematic analysis was performed to synthesize common decision factors across studies. RESULTS: We screened 1,972 abstracts and identified 7 studies for inclusion, reporting on 1,575 providers. Thematic analysis showed that referring providers consider factors related to the specialist's clinical expertise (skill, training, outcomes, and assessments), interactions between the patient and specialist (prior experience, rapport, location, scheduling, preference, and insurance), and interactions between the referring physician and specialist (personal relationships, communication, reputation, reciprocity, and practice or system affiliation). Notably, studies did not describe how providers assess clinical or technical skills. CONCLUSIONS: Referring providers rely on subjective factors and assessments to evaluate quality when selecting a specialist. There may be a role for guidelines and objective measures of quality to inform the choice of specialist by referring providers.

The Art of Science Communication-A Novel Approach to Science Communication Training.

Effective communication is a requisite skill for scientists. However, formalized training in this area is often unavailable for members of the scientific community. As one approach to combat this problem, the American Society for Biochemistry and Molecular Biology (ASBMB) developed The Art of Science Communication, an eight-week-long online course that provides facilitated instruction on how to communicate science in an oral format. The course is offered three times a year, and as of December 2017, nearly 200 individuals from all career stages have taken part in it. The course completion rate is currently 60%, a rate three to five times as high as the average for similar Massive Open Online Courses (MOOCs). Participants have indicated that taking the course has improved their ability to communicate about their research, and that the skills and lessons learned have benefited them professionally. Moving forward, we are examining approaches that will help us improve the course and expand its reach throughout the scientific community. This article details the development of the course and examines the role and potential of such training within the larger scientific community.

Tenofovir Disoproxil Fumarate Fails to Prevent HIV Acquisition or the Establishment of a Viral Reservoir: Two Case Reports.

The use of antiretrovirals as pre-exposure prophylaxis (PrEP) is highly efficacious in HIV prevention. The World Health Organization recently recommended Truvada(®) (Gilead Sciences, Inc.) or tenofovir disoproxil fumarate (TDF) for high-risk individuals, with limited data for single-agent TDF PrEP in men who have sex with men (MSM). We report two cases of TDF PrEP failure in MSM who had received long-term TDF for hepatitis B infection and had therapeutic levels of drug immediately after HIV acquisition. Rapid antiretroviral intensification at diagnosis of acute HIV infection failed to limit immune dysfunction or prevent the establishment of a viral reservoir.

Failure of daily tenofovir to prevent HIV transmission or the establishment of a significant viral reservoir despite continued antiretroviral therapy.

INTRODUCTION: Truvada is licenced for HIV-1 prevention in the USA and is available in the private sector. Tenofovir performed as well as Truvada in the PARTNERS PrEP study and is used as HIV pre-exposure prophylaxis (PreP) in some settings. The clinical efficacy of Tenofovir for PrEP outside a clinical trial is unknown. Antiretroviral therapy (ART) at acute HIV-1 infection (AHI) limits the size of the reservoir, optimizing the chance of maintaining viral control off therapy. As such ART at acute HIV infection is proposed to offer a functional cure in a minority of subjects. We present two cases where Tenofovir PrEP failed to prevent HIV acquisition and failed to limit viral reservoir. MATERIALS AND METHODS: Two individuals receiving tenofovir monotherapy for Hepatitis B monoinfection were diagnosed with AHI as defined by a negative HIV antibody test within three months of a positive HIV test following unsafe sex with casual male partners. In-depth histories were taken. Viral genotypes and Tenofovir drug levels were measured from samples taken as close to HIV seroconversion as possible and subsequent samples were analyzed for proviral Total HIV-1 DNA by qPCR. RESULTS: Patient A had received tenofovir for the preceding six years and always maintained an undetectable Hepatitis B viral load with no concerns about adherence. Two weeks preceding the positive HIV antibody test, he experienced mild symptoms (fever, pharyngitis) of HIV seroconversion. HIV status was confirmed by a repeat fourth generation HIV antibody test and by Western Blot and an HIV viral load was undetectable. Tenofovir trough level at HIV diagnosis was within normal limits. The regimen was intensified to Eviplera and a total HIV-1 DNA was 1381 copies/million CD4 T cells. Patient B received four regimens for hepatitis B treatment before starting tenofovir monotherapy in 2011 and subsequently maintained an undetectable hepatitis B viral load. After three years of tenofovir monotherapy he developed a severe symptomatic seroconversion illness and tested HIV antibody positive. The baseline HIV viral load was 103,306 copies/mL. The regimen was intensified and total HIV-1 DNA was 2746 copies/million CD4 T cells. CONCLUSIONS: Further investigation into the efficacy of tenofovir for PrEP outside a clinical trial is required. ART at AHI does not always lead to a low viral reservoir. To explore the possibility of replication incompetent virus, viral outgrowth assays are underway.

Pharmacogenetics of resistance to Cisplatin and other anticancer drugs and the role of sphingolipid metabolism.

Dictyostelium discoideum has proven to be a useful lead genetic system for identifying novel genes and pathways responsible for the regulation of sensitivity to the widely used anticancer drug cisplatin. Resistance to cisplatin is a major factor limiting the efficacy of the drug in treating many types of cancer. Studies using unbiased insertional mutagenesis in D. discoideum have identified the pathway of sphingolipid metabolism as a key regulator in controlling sensitivity to cisplatin. Using the genetic tools including directed homologous recombination and ectopic gene expression available with D. discoideum has shown how pharmacological modulation of this pathway can increase sensitivity to cisplatin, and these results have been extensively translated to, and validated in, human cells. Strategies, experimental conditions, and methods are presented to enable further study of resistance to cisplatin as well as other important drugs.

Lead genetic studies in Dictyostelium discoideum and translational studies in human cells demonstrate that sphingolipids are key regulators of sensitivity to cisplatin and other anticancer drugs.

A Dictyostelium discoideum mutant with a disruption in the sphingosine-1-phosphate (S-1-P) lyase gene was obtained in an unbiased genetic analysis, using random insertional mutagenesis, for mutants with increased resistance to the widely used cancer chemotherapeutic drug cisplatin. This finding opened the way to extensive studies in both D. discoideum and human cells on the role and mechanism of action of the bioactive sphingolipids S-1-P and ceramide in regulating the response to chemotherapeutic drugs. These studies showed that the levels of activities of the sphingolipid metabolizing enzymes S-1-P lyase, sphingosine kinase and ceramide synthase, affect whether a cell dies or lives in the presence of specific drugs. The demonstration that multiple enzymes of this biochemical pathway were involved in regulating drug sensitivity provided new opportunities to test whether pharmacological intervention might increase sensitivity. Thus it is of considerable clinical significance that pharmacological inhibition of sphingosine kinase synergistically sensitizes cells to cisplatin, both in D. discoideum and human cells. Linkage to the p38 MAP kinase and protein kinase C (PKC) signaling pathways has been demonstrated. This work demonstrates the utility of D. discoideum as a lead genetic system to interrogate molecular mechanisms controlling the sensitivity of tumor cells to chemotherapeutic agents and for determining novel ways of increasing efficacy. The D. discoideum system could be easily adapted to a high throughput screen for novel chemotherapeutic agents.

Stress-induced ER to Golgi translocation of ceramide synthase 1 is dependent on proteasomal processing.

The ceramide synthase (CerS) enzymes are key regulators of ceramide homeostasis. CerS1 is central to regulating C18 ceramide which has been shown to be important in cancer and the response to chemotherapeutic drugs. Previous work indicated that some drugs induced a novel and specific translocation of CerS1 from the endoplasmic reticulum to the Golgi apparatus. We now show that diverse stresses such as UV light, DTT, as well as drugs with different mechanisms of action induce CerS1 translocation. The stresses cause a specific cleavage of the CerS1 enzyme, and the cleavage is dependent on the action of the proteasome. Inhibition of proteasome function inhibits stress-induced CerS1 translocation, indicating that this proteolytic cleavage precedes the translocation. Modulation of protein kinase C activity shows that it plays a central role in regulating CerS1 translocation. Analysis of the C-terminus of the CerS1 protein shows that several KxKxx motifs are not involved in regulating stress induced translocation. The study suggests that diverse stresses initiate responses through different signaling pathways, which ultimately converge to regulate CerS1 localization. The data provide an increasingly detailed understanding of the regulation of this important enzyme in normal and stressed cells and support the idea that it is uniquely regulated with respect to the other CerS enzymes.

Ceramide synthase 1 is regulated by proteasomal mediated turnover.

Ceramide is an important bioactive lipid, intimately involved in many cellular functions, including the regulation of cell death, and in cancer and chemotherapy. Ceramide is synthesized de novo from sphinganine and acyl CoA via a family of 6 ceramide synthase enzymes, each having a unique preference for different fatty acyl CoA substrates and a unique tissue distribution. However, little is known regarding the regulation of these important enzymes. In this study we focus on ceramide synthase 1 (CerS1) which is the most structurally and functionally distinct of the enzymes, and describe a regulatory mechanism that specifically controls the level of CerS1 via ubiquitination and proteasome dependent protein turnover. We show that both endogenous and ectopically expressed CerS1 have rapid basal turnover and that diverse stresses including chemotherapeutic drugs, UV light and DTT can induce CerS1 turnover. The turnover requires CerS1 activity and is regulated by the opposing actions of p38 MAP kinase and protein kinase C (PKC). p38 MAP kinase is a positive regulator of turnover, while PKC is a negative regulator of turnover. CerS1 is phosphorylated in vivo and activation of PKC increases the phosphorylation of the protein. This study reveals a novel and highly specific mechanism by which CerS1 protein levels are regulated and which directly impacts ceramide homeostasis.

Global transcriptional responses to cisplatin in Dictyostelium discoideum identify potential drug targets.

Dictyostelium discoideum is a useful model for studying mechanisms of cisplatin drug sensitivity. Our previous findings, that mutations in sphingolipid metabolism genes confer cisplatin resistance in D. discoideum and in human cells, raised interest in the resistance mechanisms and their implications for cisplatin chemotherapy. Here we used expression microarrays to monitor physiological changes and to identify pathways that are affected by cisplatin treatment of D. discoideum. We found >400 genes whose regulation was altered by cisplatin treatment of wild-type cells, including groups of genes that participate in cell proliferation and in nucleotide and protein metabolism, showing that the cisplatin response is orderly and multifaceted. Transcriptional profiling of two isogenic cisplatin-resistant mutants, impaired in different sphingolipid metabolism steps, showed that the effect of cisplatin treatment was greater than the effect of the mutations, indicating that cisplatin resistance in the mutants is due to specific abilities to overcome the drug effects rather than to general drug insensitivity. Nevertheless, the mutants exhibited significantly different responses to cisplatin compared with the parent, and >200 genes accounted for that difference. Mutations in five cisplatin response genes (sgkB, csbA, acbA, smlA, and atg8) resulted in altered drug sensitivity, implicating novel pathways in cisplatin response. Our data illustrate how modeling complex cellular responses to drugs in genetically stable and tractable systems can uncover new targets with the potential for improving chemotherapy.

(Dihydro)ceramide synthase 1 regulated sensitivity to cisplatin is associated with the activation of p38 mitogen-activated protein kinase and is abrogated by sphingosine kinase 1.

Resistance to chemotherapeutic drugs often limits their clinical efficacy. Previous studies have implicated the bioactive sphingolipid sphingosine-1-phosphate (S-1-P) in regulating sensitivity to cisplatin [cis-diamminedichloroplatinum(II)] and showed that modulating the S-1-P lyase can alter cisplatin sensitivity. Here, we show that the members of the sphingosine kinase (SphK1 and SphK2) and dihydroceramide synthase (LASS1/CerS1, LASS4/CerS4, and LASS5/CerS5) enzyme families each have a unique role in regulating sensitivity to cisplatin and other drugs. Thus, expression of SphK1 decreases sensitivity to cisplatin, carboplatin, doxorubicin, and vincristine, whereas expression of SphK2 increases sensitivity. Expression of LASS1/CerS1 increases the sensitivity to all the drugs tested, whereas LASS5/CerS5 only increases sensitivity to doxorubicin and vincristine. LASS4/CerS4 expression has no effect on the sensitivity to any drug tested. Reflecting this, we show that the activation of the p38 mitogen-activated protein (MAP) kinase is increased only by LASS1/CerS1, and not by LASS4/CerS4 or LASS5/CerS5. Cisplatin was shown to cause a specific translocation of LASS1/CerS1, but not LASS4/CerS4 or LASS5/CerS5, from the endoplasmic reticulum (ER) to the Golgi apparatus. Supporting the hypothesis that this translocation is mechanistically involved in the response to cisplatin, we showed that expression of SphK1, but not SphK2, abrogates both the increased cisplatin sensitivity in cells stably expressing LASS1/CerS and the translocation of the LASS1/CerS1. The data suggest that the enzymes of the sphingolipid metabolic pathway can be manipulated to improve sensitivity to the widely used drug cisplatin.

Proteomics of canine lymphoma identifies potential cancer-specific protein markers.

PURPOSE: Early diagnosis of cancer is crucial for the success of treatment of the disease, and there is a need for markers whose differential expression between disease and normal tissue could be used as a diagnostic tool. Spontaneously occurring malignancies in pets provide a logical tool for translational research for human oncology. Lymphoma, one of the most common neoplasms in dogs, is similar to human non-Hodgkin's lymphoma and could serve as an experimental model system. EXPERIMENTAL DESIGN: Thirteen lymph nodes from normal dogs and 11 lymph nodes from dogs with B-cell lymphoma were subjected to proteomic analysis using two-dimensional PAGE separation and matrix-assisted laser desorption/ionization time-of-flight analysis. RESULTS: A total of 93 differentially expressed spots was subjected to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis, and several proteins that showed differential expression were identified. Of these, prolidase (proline dipeptidase), triosephosphate isomerase, and glutathione S-transferase were down-regulated in lymphoma samples, whereas macrophage capping protein was up-regulated in the lymphoma samples. CONCLUSIONS: These proteins represent potential markers for the diagnosis of lymphoma and should be further investigated in human samples for validation of their utility as diagnostic markers.

Sensitive cell viability assay for use in drug screens and for studying the mechanism of action of drugs in Dictyostelium discoideum.

In order to increase the effectiveness of Dictyostelium discoideum as a lead genetic model for drug discovery, a luminescence-based assay has been adapted and standardized for sensitive and rapid cell viability measurements. The applicability of the assay was demonstrated by measuring the cytotoxicity of several drugs in wild-type and mutant cells. The robustness and ease of the assay demonstrate that it can be used in high-throughput applications such as drug or mutant screens. Conclusions from these studies are applicable to evaluating cell viability assays in other systems as well.

Towards a molecular understanding of human diseases using Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is increasingly being used as a simple model for the investigation of problems that are relevant to human health. This article focuses on several recent examples of Dictyostelium-based biomedical research, including the analysis of immune-cell disease and chemotaxis, centrosomal abnormalities and lissencephaly, bacterial intracellular pathogenesis, and mechanisms of neuroprotective and anti-cancer drug action. The combination of cellular, genetic and molecular biology techniques that are available in Dictyostelium often makes the analysis of these problems more amenable to study in this system than in mammalian cell culture. Findings that have been made in these areas using Dictyostelium have driven research in mammalian systems and have established Dictyostelium as a powerful model for human-disease analysis.

Dictyostelium discoideum to human cells: pharmacogenetic studies demonstrate a role for sphingolipids in chemoresistance.

Resistance to chemotherapy is a major obstacle for the treatment of cancer and a subject of extensive research. Numerous mechanisms of drug resistance have been proposed, and they differ for different drugs. Nevertheless, it is clear that our understanding of this important problem is still incomplete, and that new targets for modulating therapy still await discovery. The attractive biology and the availability of powerful molecular techniques have made the cellular slime mold Dictyostelium discoideum, a powerful non-mammalian model for drug target discovery, and the problem of drug resistance. To understand the molecular basis of chemoresistance to the widely used drug cisplatin, both genetic and pharmacological approaches have been applied to this versatile experimental system. These studies have resulted in the identification of novel molecular pathways which can be used to increase the efficacy of cisplatin, and brought attention to the role of sphingolipids in mediating the cellular response to chemotherapeutic drugs. In the following review, we will describe the history and utility of D. discoideum in pharmacogenetics, and discuss recent studies which focus attention on the role of sphingolipids in chemotherapy and chemoresistance.

Sphingosine-1-phosphate lyase regulates sensitivity of human cells to select chemotherapy drugs in a p38-dependent manner.

Resistance to cisplatin is a common problem that limits its usefulness in cancer therapy. Molecular genetic studies in the model organism Dictyostelium discoideum have established that modulation of sphingosine kinase or sphingosine-1-phosphate (S-1-P) lyase, by disruption or overexpression, results in altered cellular sensitivity to this widely used drug. Parallel changes in sensitivity were observed for the related compound carboplatin but not for other chemotherapy drugs tested. Sensitivity to cisplatin could also be potentiated pharmacologically with dimethylsphingosine, a sphingosine kinase inhibitor. We now have validated these studies in cultured human cell lines. HEK293 or A549 lung cancer cells expressing human S-1-P lyase (hSPL) show an increase in sensitivity to cisplatin and carboplatin as predicted from the earlier model studies. The hSPL-overexpressing cells were also more sensitive to doxorubicin but not to vincristine or chlorambucil. Studies using inhibitors to specific mitogen-activated protein kinases (MAPK) show that the increased cisplatin sensitivity in the hSPL-overexpressing cells is mediated by p38 and to a lesser extent by c-Jun NH2-terminal kinase MAPKs. p38 is not involved in vincristine or chlorambucil cytotoxicity. Measurements of MAPK phosphorylation and enzyme activity as well as small interfering RNA inhibition studies show that the response to the drug is accompanied by up-regulation of p38 and c-Jun NH2-terminal kinase and the lack of extracellular signal-regulated kinase up-regulation. These studies confirm an earlier model proposing a mechanism for the drug specificity observed in the studies with D. discoideum and support the idea that the sphingosine kinases and S-1-P lyase are potential targets for improving the efficacy of cisplatin therapy for human tumors.

Sphingosine kinase regulates the sensitivity of Dictyostelium discoideum cells to the anticancer drug cisplatin.

The drug cisplatin is widely used to treat a number of tumor types. However, resistance to the drug, which remains poorly understood, limits its usefulness. Previous work using Dictyostelium discoideum as a model for studying drug resistance showed that mutants lacking sphingosine-1-phosphate (S-1-P) lyase, the enzyme that degrades S-1-P, had increased resistance to cisplatin, whereas mutants overexpressing the enzyme were more sensitive to the drug. S-1-P is synthesized from sphingosine and ATP by the enzyme sphingosine kinase. We have identified two sphingosine kinase genes in D. discoideum--sgkA and sgkB--that are homologous to those of other species. The biochemical properties of the SgkA and SgkB enzymes suggest that they are the equivalent of the human Sphk1 and Sphk2 enzymes, respectively. Disruption of the kinases by homologous recombination (both single and double mutants) or overexpression of the sgkA gene resulted in altered growth rates and altered response to cisplatin. The null mutants showed increased sensitivity to cisplatin, whereas mutants overexpressing the sphingosine kinase resulted in increased resistance compared to the parental cells. The results indicate that both the SgkA and the SgkB enzymes function in regulating cisplatin sensitivity. The increase in sensitivity of the sphingosine kinase-null mutants was reversed by the addition of S-1-P, and the increased resistance of the sphingosine kinase overexpressor mutant was reversed by the inhibitor N,N-dimethylsphingosine. Parallel changes in sensitivity of the null mutants are seen with the platinum-based drug carboplatin but not with doxorubicin, 5-fluorouracil, and etoposide. This pattern of specificity is similar to that observed with the S-1-P lyase mutants and should be useful in designing therapeutic schemes involving more than one drug. This study identifies the sphingosine kinases as new drug targets for modulating the sensitivity to platinum-based drugs.

Proteomic analysis to identify breast cancer biomarkers in nipple aspirate fluid.

PURPOSE: Proteomic analysis of breast nipple aspirate fluid (NAF) holds promise as a noninvasive method to identify markers of breast cancer. The objectives of the study were to: (a) describe the NAF proteome, (b) identify candidate markers of breast cancer in NAF by using proteomic analysis, and (c) validate the markers identified by using a quantitative, high-throughput ELISA analysis. EXPERIMENTAL DESIGN: For proteome analysis, NAF proteins from a single subject without breast cancer were separated by two-dimensional PAGE and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectometry identification. A total of 41 different proteins were identified, 25 of which were known to be secreted. To identify breast cancer markers, we separated 20 NAF samples (10 normal, 10 cancer) by two-dimensional PAGE. Three protein spots were detected that were up-regulated in three or more cancer samples. These spots were identified to be gross cystic disease fluid protein (GCDFP)-15, apolipoprotein D (apoD), and alpha1-acid glycoprotein (AAG). To validate these three potential biomarkers, 105 samples (53 from benign breasts and 52 from breasts with cancer) were analyzed using ELISA. RESULTS: Among all of the subjects, GCDFP-15 levels were lower (P <0.001) and AAG levels were higher (P=0.001) in breasts with cancer. This was also true in premenopausal (GCDFP-15, P=0.011; AAG, P=0.002) but not in postmenopausal women. GCDFP-15 levels were lowest (P=0.003) and AAG levels highest (P <0.001) in women with ductal carcinoma in situ (DCIS). Menopausal status influenced GCDFP-15 and AAG more in women without breast cancer than in women with breast cancer. apoD levels did not correlate significantly with breast cancer. CONCLUSIONS: Our study revealed that the NAF proteome, as defined by two-dimensional PAGE, consists of a limited number of proteins, and that the expression of AAG and GCDFP-15 correlates with disease presence and stage.

Sphingosine-1-phosphate plays a role in the suppression of lateral pseudopod formation during Dictyostelium discoideum cell migration and chemotaxis.

Sphingosine-1-phosphate (S-1-P) is a bioactive lipid that plays a role in diverse biological processes. It functions both as an extracellular ligand through a family of high-affinity G-protein-coupled receptors, and intracellularly as a second messenger. A growing body of evidence has implicated S-1-P in controlling cell movement and chemotaxis in cultured mammalian cells. Mutant D. discoideum cells, in which the gene encoding the S-1-P lyase had been specifically disrupted by homologous recombination, previously were shown to be defective in pseudopod formation, suggesting that a resulting defect might exist in motility and/or chemotaxis. To test this prediction, we analyzed the behavior of mutant cells in buffer, and in both spatial and temporal gradients of the chemoattractant cAMP, using computer-assisted 2-D and 3-D motion analysis systems. Under all conditions, S-1-P lyase null mutants were unable to suppress lateral pseudopod formation like wild-type control cells. This resulted in a reduction in velocity in buffer and spatial gradients of cAMP. Mutant cells exhibited positive chemotaxis in spatial gradients of cAMP, but did so with lowered efficiency, again because of their inability to suppress lateral pseudopod formation. Mutant cells responded normally to simulated temporal waves of cAMP but mimicked the temporal dynamics of natural chemotactic waves. The effect must be intracellular since no homologs of the S-1-P receptors have been identified in the Dictyostelium genome. The defects in the S-1-P lyase null mutants were similar to those seen in mutants lacking the genes for myosin IA, myosin IB, and clathrin, indicating that S-1-P signaling may play a role in modulating the activity or organization of these cytoskeletal elements in the regulation of lateral pseudopod formation.

Overexpression of sphingosine-1-phosphate lyase or inhibition of sphingosine kinase in Dictyostelium discoideum results in a selective increase in sensitivity to platinum-based chemotherapy drugs.

The efficacy of the chemotherapy drug cisplatin is often limited due to resistance of the tumors to the drug, and increasing the potency of cisplatin without increasing its concentration could prove beneficial. A previously characterized Dictyostelium discoideum mutant with increased resistance to cisplatin was defective in the gene encoding sphingosine-1-phosphate (S-1-P) lyase, which catalyzes the breakdown of S-1-P, an important regulatory molecule in cell function and development and in the regulation of cell fate. We hypothesized that the increased resistance to cisplatin was due to an elevation of S-1-P and predicted that lowering levels of S-1-P should increase sensitivity to the drug. We generated three strains that stably overexpress different levels of the S-1-P lyase. The overexpressor strains have reduced growth rate and, confirming the hypothesis, showed an expression-dependent increase in sensitivity to cisplatin. Consistently, treating the cells with D-erythro-N,N,-dimethylsphingosine, a known inhibitor of sphingosine kinase, increased the sensitivity of mutant and parent cells to cisplatin, while addition of exogenous S-1-P or 8-Br-cyclic AMP made the cells more resistant to cisplatin. The increased sensitivity of the overexpressors to cisplatin was also observed with the cisplatin analog carboplatin. In contrast, the response to doxorubicin, 5-flurouracil, or etoposide was unaffected, indicating that the involvement of the sphingolipid metabolic pathway in modulating the response to cisplatin is not part of a global genotoxic stress response. The augmented sensitivity to cisplatin appears to be the result of an intracellular signaling function of S-1-P, because D. discoideum does not appear to have endothelial differentiation growth (EDG/S1P) receptors. Overall, the results show that modulation of the sphingolipid pathway at multiple points can result in increased sensitivity to cisplatin and has the potential for increasing the clinical usefulness of this important drug.