What were the critical steps in the development of ATRA and arsenic as treatments for APL? Researchers in Shanghai tell the story and look to the future Acute promyelocytic leukemia (APL) was first identified as a distinct subtype of acute myeloid leukemia in 1957 by Leif Hillestad. It is called M3 in the French–American–British classification, with a variant type referred to as microgranular (M3v in the French–American–British nomenclature) Apoptosis: A genetically determined process of cell death in which the cell uses specialized cellular machinery to kill itself and is then eliminated by phagocytosis or by shedding.Caspase: A family of cysteine proteases with aspartate specificity that are essential intracellular death effectors.Disseminated intravascular coagulation: A hemorrhagic disorder that occurs following the uncontrolled activation of clotting factors and fibrinolytic enzymes throughout small blood vessels, resulting in depletion of clotting factors and tissue necrosis and bleeding.Fibrinogenopenia: A decrease in concentration of fibrinogen in the blood.Granulocyte: Terminally differentiated myelocyte or polymorphonuclear white blood cell (as a basophil, eosinophil, or neutrophil) with granule-containing cytoplasm.Ligand-inducible transcription factors: Transcription factors that structurally have domains associated with DNA binding and ligand (hormone) recognition. When binding to its specific ligand, the transcription factor initiates a series of conformational changes and interacts efficiently with its specific DNA response element to recruit components of the transcriptional machinery.Nuclear receptor superfamily: One of the most abundant classes of transcriptional regulators including receptors for steroid hormones (e.g., estrogens, glucocorticoids, and vitamin D3), RAs, thyroid hormones, and so on. These transcription factors regulate diverse functions such as homeostasis, reproduction, development, and metabolism in animals.Promyelocyte: Granule-containing cell in bone marrow that is in an intermediate stage of development between myeloblasts and myelocytes and that gives rise to a granulocyte.Proteasome: Proteolytic complex that degrades cytosolic and nuclear proteins.Sumoylation: Post-translational modification of proteins by the small ubiquitin-like modifier SUMO.Ubiquitin: A chiefly eukaryotic protein that when covalently bound to other cellular proteins marks them for proteolytic degradation.APL accounts for 10%–15% of all cases of acute myeloid leukemia, with several thousand new cases diagnosed worldwide each year. Before the advent of differentiation therapy, APL was treated with anthracycline-based chemotherapy with a complete remission rate of 60%–76% and a 5-year event-free survival rate of 23%–35% [Failure to differentiate terminally characterizes most, if not all, cancer cells of every origin. Whether the induction of differentiation could be a treatment strategy for cancers was hotly debated for decades before the advent of differentiation therapy.cis RA in people with refractory or relapsed APL [cis RA was ineffective in treating APL An important discovery of the early 1970s was that myeloid leukemic cells could be reprogrammed to resume normal differentiation and to become non-dividing mature granulocytes or macrophages as a result of stimulation by various cytokines ,4. Basedpsed APL ,9,10, bu2/day). The result was dramatic: 23 patients (95.8%) went into complete remission (CR) without developing bone marrow hypoplasia or abnormalities of clotting. The remaining one patient achieved CR when chemotherapy was added Beginning in the early 1980s, the Shanghai Institute of Hematology conducted a series of experiments on differentiation therapy for APL. These experiments showed that all-trans RA (ATRA) could induce terminal differentiation of HL-60, a cell line with promyelocytic features, and fresh leukemic cells from patients with APL. These intriguing results were the impetus for a clinical trial. Twenty-four patients with APL were treated with ATRA , yellow arsenic , and white arsenic, or arsenic trioxide (As2O3) Arsenic is a common, naturally occurring substance that exists in organic and inorganic forms in nature. The organic arsenicals consist of an arsenic atom in its trivalent or pentavalent state linked covalently to a carbon atom. There are three inorganic forms of arsenic: red arsenic  and induces partial differentiation at low concentrations (0.1 × 10−6 to 0.5 × 10−6 M). The rapid modulation and degradation of the PML-RARa oncoprotein by arsenic trioxide could contribute to these two effects Studies have shown that arsenic trioxide exerts dose-dependent dual effects on APL cells—it induces apoptosis (programmed cell death) preferentially at relatively high concentrations (0.5 × 10To understand how ATRA and arsenic compounds act at the molecular level in treating APL, it is first important to understand the role of the PML-RARa fusion protein in the pathogenesis of APL.−9–10−8 M) of retinoids  and retinoid X receptors (RXRs). These belong to the steroid/thyroid/retinoid nuclear receptor superfamily of ligand-inducible transcription factors. Both RAR and RXR families consist of three subtypes: α, β, and γ etinoids .More than 95% of patients with APL have the tq22;q21) translocation. This results in the fusion of the RARα gene on 17q21 and the promyelocytic leukemia (PML) gene on 15q22, which generates a PML-RARaα fusion transcript [ translocPML-RARa may affect transcription in other pathways including those in which the transcription factor AP1 and interferon-responsive factors are involved. PML-RARα also binds to promyelocytic leukemia zinc finger (PLZF) protein and potentially affects its functions  −7–10−6 M) exert their effects through targeting the PML-RARα oncoprotein, triggering both a change in configuration and degradation of the oncoprotein and the activation of transcription, leading to differentiation. Cleavage of the PML-RARα fusion protein by caspases at residue D522 has been shown in APL cells induced to differentiate by ATRA ATRA and arsenic trioxide degrade and cleave the PML-RARα oncoprotein. Although we now have a good understanding of the molecular mechanisms underlying ATRA in differentiation therapy for APL, these mechanisms were shown long after the identification of the efficacy of this drug in treating the disease. Now it is well established that pharmacological concentrations of ATRA , and this difference persisted after consolidation therapy (p < 0.05). Notably, all 20 patients in the combination group remained in CR whereas seven of 37 cases treated with monotherapy relapsed (p < 0.05) after a follow-up of 8–30 months (median, 18 months).Since ATRA and arsenic trioxide degrade the PML-RARa oncoprotein via different pathways, and since studies in animal models have shown synergic effects of both drugs in prolonging survival or even eliminating the disease ,35, the It seems that a combination of ATRA and arsenic trioxide for remission and maintenance treatment of APL produces better results than either of the two drugs used alone, in terms of the time required to achieve CR and the length of disease-free survival. We hope that the use of three treatments—ATRA, arsenic trioxide, and chemotherapy—will ultimately make APL a curable human acute myeloid leukemia The story of ATRA in the treatment of APL shows that by targeting the molecules critical to the pathogenesis of certain diseases, cells can be induced to return to normal. Differentiation therapy is therefore a practical method of treating human cancer that has shown consistent effectiveness in trials. The clarification of the underlying molecular abnormalities of APL is an example of the benefits of a close collaboration between bench and bedside, and is necessary for our understanding of the mechanisms of ATRA in differentiation therapy. It is clearly important to elucidate the molecular and cellular basis of a particular cancer if we are to further develop mechanism-based target therapies.The sequencing of the human genome and ongoing functional genomic research are now accelerating the dissection of disease mechanisms and identification of therapeutic targets. This in turn may facilitate the screening of promising treatments. What we learn from developing curative treatment approaches to APL may help to conquer other types of leukemias and cancers.


Increasing prevalence of obesity and disorders associated with sedentary living constitute a major global public health problem. While previous evaluations of interventions to increase physical activity have involved communities or individuals with established disease, less attention has been given to interventions for individuals at risk of disease.ProActive aims to evaluate the efficacy of a theoretical, evidence- and family-based intervention programme to increase physical activity in a sedentary population, defined as being at-risk through having a parental family history of diabetes. Primary care diabetes or family history registers were used to recruit 365 individuals aged 30–50 years, screened for activity level. Participants were assigned by central randomisation to three intervention programmes: brief written advice (comparison group), or a psychologically based behavioural change programme, delivered either by telephone (distance group) or face-to-face in the family home over one year. The protocol-driven intervention programme is delivered by trained facilitators, and aims to support increases in physical activity through the introduction and facilitation of a range of self-regulatory skills . The primary outcome is daytime energy expenditure and its ratio to resting energy expenditure, measured at baseline and one year using individually calibrated heart rate monitoring. Secondary measures include self-report of individual and family activity, psychological mediators of behaviour change, physiological and biochemical correlates, acceptability, and costs, measured at baseline, six months and one year. The primary intention to treat analysis will compare groups at one-year post randomisation. Estimation of the impact on diabetes incidence will be modelled using data from a parallel ten-year cohort study using similar measures.ProActive is the first efficacy trial of an intervention programme to promote physical activity in a defined high-risk group accessible through primary care. The intervention programme is based on psychological theory and evidence; it introduces and facilitates the use of self-regulatory skills to support behaviour change and maintenance. The trial addresses a range of methodological weaknesses in the field by careful specification and quality assurance of the intervention programme, precise characterisation of participants, year-long follow-up and objective measurement of physical activity. Due to report in 2005, ProActive will provide estimates of the extent to which this approach could assist at-risk groups who could benefit from changes in behaviours affecting health, and inform future pragmatic trials. This trial addresses the rise in the burden of disease associated with sedentary living: a major public health problem. Physical inactivity accounts for up to 11.7% of all deaths in developed countries [Reversal of this trend will require not only public health programmes to increase activity at societal level, but also interventions to help high-risk individuals increase physical activity and maintain beneficial activity patterns . This is2 [The study targets people with a parental family history of Type 2 diabetes and a sedentary lifestyle, who constitute a clearly identifiable high-risk population . A consi2 , and pro2 ,6.Most trials have evaluated increasing physical activity in the context of established disease. The few published trials of primary prevention in high-risk groups have methodological limitations. They have mainly evaluated brief interventions to increase exercise in the general population delivered through primary care practitioners -9, oftenThree trials among individuals with impaired glucose tolerance in China , FinlandProActive intervention programme included a review of psychological theories and evidence, through systematic reviews [Many of the available trials evaluated interventions that were not explicitly based on psychological theory and evidence, and did not specify clearly which behaviour change techniques were applied by the providers -20. In a reviews ,20 and e reviews  was seleet al., 2004. A causal modelling approach to the development of theory-based behaviour change programmes for trial evaluation. Submitted).A range of behaviour change techniques with evidence for their effectiveness was used to bridge the gap between intention and action: goal setting and review, action planning, use of prompts, self-monitoring, and reinforcement ,18,23,25ProActive.Major challenges in promoting physical activity are maintenance of behaviour change, and the avoidance of drop out rates that can approach 50% . ReviewsProActive is to determine the effects of a theoretical- and evidence-based intervention programme on objectively measured physical activity after one year, in sedentary individuals at risk of diabetes and related metabolic abnormalities due to their family history. Three questions are posed:The primary objective of 1. Behaviour change: Can an innovative approach to increasing physical activity achieve clinically important change in this behaviour when offered to a group at increased risk of diabetes?2. Disease impact: If so, what is the potential for the changes in behaviour achieved in mid-life to reduce the incidence of diabetes in later life?3. Dose finding: How does delivery of the approach, at two levels of intensity, affect acceptability, efficacy and costs?The trial will estimate the extent to which physical activity and its key psychological mediators are altered by the intervention programme, and assess its acceptability to this high-risk group. It will document the extent to which behaviour change is associated with reduction in weight gain and improvement in physiological and biochemical correlates, and will model the potential impact of the intervention on future risk of diabetes.Intensive, face-to-face interventions may not be a feasible health service model, and there is some evidence that less intensive, continuous support may be as effective ,23. The ProActive is a four-year study with a complex randomised trial design [l design , with ceThe study design and patient flows (achieved at recruitment closure, October 2003, and projected to end of study) are shown in Figure The study is explanatory in design, and the quality-assured intervention programmes are delivered by carefully trained and supervised family health facilitators with experience of working in primary care or the community, and backgrounds in health promotion, dietetics and nursing.The study is set in urban, suburban and rural Cambridgeshire, Essex and West Suffolk, England, in the homes of participants and their families. The study population consists of offspring of people with Type 2 diabetes, aged 30–50 years, without a diagnosis of diabetes, and not considered very active based on self-report at the start of the study (see below). This age range defines a group at risk of weight gain ,32. Any Once the relevant ethical and PCT approval had been obtained, 53 practice teams in the locality were approached by letter, inviting them to take part in the study, and highlighting the reimbursement of all costs involved. Personalised letters were sent to the practice manager (who we asked to collate responses and reply using a reply slip and Freepost envelope), all partners and nursing staff. Included with each letter was a brief summary of the study and a Research Information Sheet for Practices (RISP) form . If no rInitially participants were recruited through their parents; patients with Type 2 diabetes on the diabetes registers of 20 practices ('recruitment method 1'). Patients were written to by their general practitioner, with a description of the study, and asked to provide contact information for any offspring aged 30–50 years, living locally. Consent was also sought for the practice to pass the contact details of the offspring to the research team so that they could invite the offspring directly into the study. Piloting demonstrated feasibility and acceptability of the method, and one reminder was sent after three weeks if no reply was received.From 20 practices, 2631 patients were approached and 2025 (77%) replied, yielding 1238 potentially eligible offspring who were invited to take part in the study. The ratio of approximately one potentially eligible offspring to two patients with diabetes was half our pilot projections, so to increase recruitment we developed a second recruitment approach ('recruitment method 2'). This approach recruited potential participants with a recorded family history of diabetes directly from practices with family history registers, and was feasible in seven of the 20 practices. General practitioners wrote to all patients aged 30–50 years with a recorded family history of diabetes, enclosing a study information sheet, and asking those willing to complete and return to the practice a questionnaire to determine which family member(s) had diabetes, and of which type. Consent was sought for this information and contact details to be passed on to the research team. Using this method, with again one reminder letter, 1340 patients were written to, and 896 (67%) responses were received, with 283 patients interested and eligible. Both recruitment approaches provided 1521 potential trial participants. Practitioners used their discretion in applying both approaches to the exclusion of patients who were physically or mentally unwell.Potential participants recruited by both methods were next written to by the research team with full information about the study and a screening activity questionnaire, describing occupational and leisure activity, based on published questionnaires ,36, to eTo fulfil measurement requirements participants had to be able to walk briskly, without help, on the flat for 15 minutes. Participants also had to live within reach of the measurement centre and the Family Health Facilitators; defined as a 30-minute average travel time from the study co-ordination centre. Other exclusion criteria included individuals with serious physical or psychiatric illness limiting programme involvement; people with life issues interfering with the study; those known to be pregnant or have diabetes before baseline measurement; and those planning to move away. As shown in Figure ProActive. Brief details of the trial were sent, together with a request for feedback if the practitioner had any concerns about the offspring's participation, or about the safety of the facilitators making home visits.Eligible offspring were registered with general practitioners in the Eastern Region of the UK. Prior to both baseline measures and randomisation, these doctors were individually informed about their registered patients' intention to participate in Randomisation was carried out centrally by the trial statistician, using a partial minimisation procedure that dynamically adjusted the randomisation probabilities in order to balance important covariates; body mass index, sex, age, physical activity , family size, and behavioural intentions. Randomisation thus used baseline measures. Thirty-two pairs of siblings and two sibling-triples were cluster randomised to the same study group to avoid contamination, and the remaining 295 participants (81%) were individually randomised. Overall, 365/465 (78%) of those eligible went forward to randomisation.At baseline and the end of the study, all participants attend the study centre at either Ely or Cambridge for questionnaires, physiological and anthropometrical measures, and venesection. At six months, psychological and self-reported physical activity data are collected by postal questionnaires. Measures relating to the intervention programme evaluation are collected by the facilitators during the intervention, and we assess reported use of self-regulatory strategies by participants to increase their activity levels at six and twelve months.In similar primary care based trials we have achieved attrition rates of 30% or less ,39, and Maximising retention is an important issue, particularly as the comparison group do not benefit from regular contact with a facilitator. At recruitment, the introductory leaflets for all three arms emphasised the importance of follow-up, irrespective of treatment group. Participants who drop out of the intervention programme are contacted by a principal investigator, and offered an opportunity to give feedback and to confirm drop out from the intervention programme only, or from trial measurement as well.The distribution of measures across baseline, six-month and one-year follow-up are shown in Table 2/kg/body weight) is measured by indirect calorimetry during a submaximal graded treadmill exercise test, and maximal cardiorespiratory fitness (VO2max) is estimated using predicted maximal heart rate (i.e. 220 minus age) [Oxygen uptake (ml Onus age) ,42.Self-report measures of well-being and quality of life include subjective health and energy (SF-36) , anxietyThe frequency and severity of physical activity related injury is assessed by study questionnaire at one year.Psychological mediators of physical activity include intention to increase activity over the next year, and its predictors . These key measures have been developed for the study following the recommendations of Ajzen . PhysiolProActive intervention programme to have an impact on health service costs in the short term, we are monitoring health service utilisation  in the last 20% of participants recruited to the study.The economic analysis will explore the impact of a physical activity intervention programme on NHS costs. As the study is explanatory in design, we will not conduct a full cost-effectiveness analysis, but aim to provide a cost-description of the delivery of the intervention programmes. We are measuring the costs of delivering the 'face-to-face' and 'distance' intervention programmes via family health facilitators. These costs primarily comprise the training of facilitators, educational materials, travel, and the time that facilitators spend contacting and visiting families (including cancelled visits). Travel costs and contact time are recorded by the facilitators for every trial participant. The cost of facilitator time will be based on national average salaries, employment costs, qualifications, overheads and indirect costs . AlthougProActive are cardiovascular and musculoskeletal events associated with the laboratory procedures of treadmill exercise testing and injuries sustained as a consequence of increasing physical activity in everyday life. The cardiorespiratory fitness test used in this study is submaximal, and only undertaken following extensive screening procedures. If a participant exhibits a positive Rose angina questionnaire [The primary safety concerns for participants in ionnaire , a positionnaire  or an abRanges for acceptable results are set for all clinical measures. If these are exceeded, the information is sent to the general practitioner, and the participant informed and advised to consult.As the intervention programme is based on participants' own preferred activities, and emphasises small achievable goals set by the participants, the risk of excess injury is small. Group information about injury will be reported.Participants previously unaware of their familial risk of diabetes may experience anxiety related to awareness of their increased risk status. This is considered in facilitator training, and measures of anxiety, worry about diabetes and perceived vulnerability are included (see above).Physiological and anthropometric measures are made in two centres by observers unaware of individuals' group allocation. Biochemical measures are made in one laboratory with established quality assurance systems. Randomisation was undertaken by the trial statistician, independently of the trial co-ordination team, and the data entry team are unaware of study group.The administrative database (participant information), dayPAR values and blood test results are managed in-house, with the latter being double entered. Numeric fields have limiters set so that values outside a defined range cannot be entered. Additionally, any blood results outside the 'normal range' are flagged for confirmation of value. Random checks on administrative data are performed regularly, checking the data on the database against paper records and correcting any errors found.Double data entry of all anthropometric and questionnaire measures is undertaken by an experienced, independent agency, blind to study group (Wyman Dillon Research and Data Management, Bristol, UK). In addition, random checks are applied as described above.The family health facilitator contacts participants randomised to the 'face-to-face' and 'distance' interventions, and arranges a home interview including family members. At this introductory interview, personal reasons for increasing physical activity are elicited and reinforced, family participation is encouraged, and the relationships between physical activity, weight gain and prevention of Type 2 diabetes are explained and discussed.In the 'face-to-face' arm this is followed by four visits and two brief support telephone calls over five months. During these interactions the participant and willing family members learn strategies to increase physical activity, for instance selecting activities that they enjoy doing, setting achievable goals, defining action plans, self-monitoring, self-reinforcement and relapse prevention. Pedometers are available for self-monitoring among participants who have chosen walking as their goal. A key difference between this intervention and others currently under evaluation (e.g. ACT) is that there is no absolute target for physical activity defined at the outset. Family members are encouraged to make gradual and continuous increases in their activity, as much as they feel able to, on the understanding that all increases, if maintained, are beneficial. Follow-up continues by monthly telephone calls up to one year, to discuss any difficulties in applying the strategies, and to encourage family members to increase activity further.In the 'distance' arm, following the introductory meeting the intervention programme is delivered by six telephone calls over five months, and then monthly by post up to one year, with content similar to the 'face-to-face' arm. During the phone calls the facilitators encourage the participants to involve family members. Visits and telephone calls take approximately one hour and 45 minutes, respectively.An arm-specific introductory leaflet is used, but otherwise materials are the same for the face-to-face and distance arms. All introductory leaflets include text to encourage retention in the trial. In the comparison arm the leaflets offer brief advice on the benefits of physical activity. Participants in the intervention programme arms are given an educational manual describing the strategies that participants are encouraged to use to increase their habitual activity in a step by step fashion.Various mechanisms are used to promote the fidelity of delivery of the intervention programme to the underlying psychological theories and intervention programme protocols. A detailed training manual and protocols for each contact were developed, and a Training Officer appointed. Facilitators attended a five day phased course in psychological theories, behaviour change techniques and experiential training in techniques, with six half-days initially, followed by refresher sessions at six months and continuing supervised practice by a clinical psychologist and through peer-appraisal. Facilitators complete a checklist for the introduction of and mastery of self-regulatory strategies by the participant after each contact, and monitor intervention programme attendance and drop-out for each participant.An assessment of adherence by facilitators to the behaviour change techniques specified in the protocols was conducted among a random sample of 27 participants, using reliable coding frames and transcripts of the sessions. The intervention programme evaluation includes: an assessment of the frequency of meetings and telephone calls, proportion of progress reports and postcards sent and progress reports returned, satisfaction with the intervention programme, reported use of self-regulatory strategies by participants at six months and one year, and drop-outs at one year.The sample size calculation was initially based on physical activity level (PAL), the ratio of total energy expenditure to estimated basal metabolic rate ,41, and Main analyses will be at one year, comparing combined 'face-to-face' and 'distance' versions of the intervention programme with 'brief advice', comparing 'face-to-face' with 'distance' modes of the intervention programme, and estimating the difference between each intervention programme group and 'brief advice' to inform a larger pragmatic trial. Analysis by intention-to-treat will retain individuals within their randomised group regardless of participation. Comparisons will involve an adjustment for baseline physical activity and other variables used in the randomisation. We will undertake sensitivity analyses, assuming a range of potential outcomes for non-completers, informed by available baseline and interim data on non-completers. Non-completers will have multiple data imputed with a 'missing at random' assumption and with sensitivity analyses to represent optimistic and pessimistic scenarios for drop out. Clustering effects by family will be estimated for the primary outcome.A secondary 'dose-response' analysis will use all three randomised groups, over baseline, six months and one year. A 'per protocol' analysis will also be undertaken among those completing the intervention programme.The incremental cost of delivering the 'face-to-face' intervention programme will be compared to the 'distance' and 'brief advice' groups.Stage 1) The trial will provide evidence on the relationship between observed behaviour change, weight change, and biochemical and physiological correlates. Modelling is facilitated by reference to the Ely Cohort; a prospective population cohort study that began in 1990 and involved 1122 people without known diabetes [ProActive.diabetes . MeasureStage 2) Using models based on past cohort data, the influence of behaviour change on future diabetes incidence [ncidence ,41,51,52Stage 3) We will undertake sensitivity analyses on the projections at Stage 2, using a range of plausible assumptions about how behaviour change might affect other risk factors and hence indirectly influence future diabetes risk.ProActive is the first efficacy trial of physical activity promotion in a defined high-risk group accessible through primary care, evaluating an intervention programme based on theory and evidence. It supports increases in informal activity, through the introduction and facilitation of self-regulatory strategies with regular reinforcement by the facilitator. Due to report in 2005, ProActive has the potential to make substantial contributions to understanding the extent to which such approaches could assist the wide range of at risk groups who could benefit most from increasing their physical activity.The trial team brings together expertise in the epidemiology of diabetes  with intet al., 2004. A causal modelling approach to the development of theory-based behaviour change programmes for trial evaluation. Submitted). This will enable replication and further strengthening of effective intervention steps, as well as development of theory.In terms of the intervention itself, careful measurement along the hypothesised causal path from cognition, through self-reported behaviours to energy expenditure, will enable testing of the application of the Theory of Planned Behaviour in this setting, and of the relationship between the everyday activities that the programme has as its focus and the objectively measured physical activity VOThe authors declare that they have no competing interests.ALK, NW, SG, SS, WH, DS, – Principal InvestigatorsTP – Trial StatisticianKW – Trial Co-ordinatorWill H – Trial EconomistUE – Physical activity measurementAll authors read and approved the final manuscript. ALK is the paper guarantor.The pre-publication history for this paper can be accessed here:


In most recent large efficacy trials of barrier contraceptive methods, a high proportion of participants withdrew before the intended end of follow-up. The objective of this analysis was to explore characteristics of participants who failed to complete seven months of planned participation in a trial of spermicide efficacy.Trial participants were expected to use the assigned spermicide for contraception for 7 months or until pregnancy occurred. In bivariable and multivariable analyses, we assessed the associations between failure to complete the trial and 17 pre-specified baseline characteristics. In addition, among women who participated for at least 6 weeks, we evaluated the relationships between failure to complete, various features of their first 6 weeks of experience with the spermicide, and characteristics of the study centers and population.Of the 1514 participants in this analysis, 635 (42%) failed to complete the study for reasons other than pregnancy. Women were significantly less likely to complete if they were younger or unmarried, had intercourse at least 8 times per month, or were enrolled at a university center or at a center that enrolled fewer than 4 participants per month. Noncompliance with study procedures in the first 6 weeks was also associated with subsequent early withdrawal, but dissatisfaction with the spermicide was not. However, many participants without these risk factors withdrew early.Failure to complete is a major problem in barrier method trials that seriously compromises the interpretation of results. Targeting retention efforts at women at high risk for early withdrawal is not likely to address the problem sufficiently. Retention of participants has been a consistent problem in clinical studies of barrier contraceptive methods. For example, in six large studies of condoms, diaphragms, and spermicides conducted in the past decade, more than 30% of the participants failed for reasons other than pregnancy to complete the intended six months or six menstrual cycles of follow-up -6 Such hIssues regarding the design of barrier method studies have become increasingly important to researchers and public health scientists since the onset of the HIV epidemic because of the urgent need for methods to prevent this disease and other sexually transmitted infections. Numerous new barrier contraceptive methods and microbicides are currently in various stages of development and testing. Devising effective approaches to maximize retention in these studies will be critical.In this analysis, we used data from a large, recently completed randomized trial of the efficacy and safety of five spermicide products to determine whether we could identify specific subgroups of participants who were at particular risk for failure to complete the trial. Our goal was to provide information that might assist in the development of targeted approaches to improve follow-up in future trials.The primary purpose of this randomized trial was to estimate and compare the probability of pregnancy during six months of typical use of five nonoxynol-9 spermicide products. Safety, acceptability, and product use were additional specified outcomes. The trial was conducted at 14 sites in the United States between June 1998 and August 2002. The study was approved by the institutional review boards at each site and at Family Health International. All participants signed written informed consent forms before enrollment.A full description of the trial procedures has been published previously . In brieFollow-up visits were scheduled at 4, 17, and 30 weeks after admission. Each participant was also asked to return to the study site if she wished to discontinue use of the spermicide. At each visit, the participant was interviewed, and a urine pregnancy test was done. At the 4-week and final visits, she completed a seven-page acceptability questionnaire. A pelvic examination was performed at the final visit and at other visits as indicated. Colposcopy substudy participants had a vaginal colposcopy at each follow-up visit. Each participant was asked to do a pregnancy test at home 2, 10, and 23 weeks after admission and to telephone the site with the result. If a participant missed a scheduled contact, study procedures required that staff make at least four attempts to contact her by at least two different modalities (telephone, mail, etc.) If they could not contact her directly, staff were to try to reach her through an alternate contact person identified by the participant at admission. Compensation for completion of all scheduled visits in the primary study ranged from $120 to $400 at the 14 study sites; at most sites, the amount was divided evenly among the separate visits.In this analysis, we included all randomized participants except for 22 who were discovered to have been pregnant at admission and who therefore contributed no data to the primary analysis. We classified each of the remaining 1514 participants as having completed the study if she considered the spermicide to be her primary contraceptive method for at least 183 days after randomization, or she became pregnant before she stopped relying on it. Otherwise, she was classified as having failed to complete the study. We assigned each participant's last day in the analysis as the earliest of the following dates: the estimated date of fertilization of a pregnancy; the date she was last known to have been relying primarily on the assigned spermicide for contraception; the latest date her pregnancy status could be reliably determined; and 183 days after randomization. These rules were the same as those used in the prior primary pregnancy analyses .We assessed the associations between failure to complete and 17 baseline factors of interest, which were prespecified before the analysis. Among the subset of participants who were in the analysis for at least 6 weeks, we examined the associations between final status category and various factors that characterized their experience during the first 6 weeks in the study. Factors were categorized in part to ensure substantial numbers of participants in each level. Hypotheses about the effects of factors on completion status were tested using chi square tests, Fisher's exact tests, Mantel Haenszel tests. Parameters estimated by multivariable logistic regressions were tested using Wald tests. We included factors in regression models if they were associated with the outcome  in bivariable analyses. None of the included factors were highly correlated. In both bivariable and multivariable analyses, a p-value of <0.05 was considered to indicate a significant association.Of the 1514 participants in this analysis, 635 (42%) failed to complete the study for reasons other than pregnancy. The proportion who withdrew early at each of the 14 study sites ranged from 17% to 83%. Only 3 centers had completion rates ≥65%. Forty nine participants (8% of those who withdrew) were discontinued by the site investigator because of a concern about their safety , staff error, or closure of the trial at the study site  relative risk of failure to complete the trial Table . FactorsOf the 1095 participants who contributed more than 6 weeks to the analysis, those who in their first 6 weeks were not compliant with follow-up visits, coital diary completion, or use of the spermicide during sex were significantly less likely than others to complete the study Table . HoweverIn analyzing data from longitudinal studies, researchers commonly assume that the experience of participants who withdraw early, had they stayed in the study, would have been similar to the experience of those who completed. However, this assumption is generally impossible to confirm and is often implausible. If the assumption is false, the study findings may substantially misrepresent the likelihood of the outcome in the study population. If the degree of misrepresentation is not consistent across study groups, comparisons could be seriously biased. Indeed, some expert epidemiologists have suggested that a trial with losses of greater than 20% of the participants "would be unlikely to successfully withstand challenges to its validity" ,9.Our study, like other recent barrier contraceptive method studies, did not even approach this standard: 42% of our enrolled participants did not complete the trial. Furthermore, the participants who failed to complete were different in key ways from those who did – they reported significantly more frequent coitus at baseline, and they also were more likely to be younger, unmarried, and poorly compliant with study procedures and method use in the first few weeks after admission. All of these characteristics were associated to some extent with elevated risk of pregnancy in our population , which sClearly, increased attention to preventing this problem in future studies is imperative. In performing this analysis, our intention was to explore the potential impact of focusing retention efforts on participants with characteristics that are associated with failure to complete. However, although we did find some factors that were significantly associated with early withdrawal, none was highly predictive; that is, many participants without these factors failed to complete the study, and many with these factors did complete. Therefore, applying special efforts only to the high-risk participants would not likely have been sufficient to raise completion rates to desirable levels. In future trials, aggressive follow-up measures should be instituted universally. Such efforts might include assigning individual "case-workers" to participants, using novel means for communicating with the participants, such as pagers, conducting visits at participants' homes or at other locations convenient for them, providing specific reimbursement for expenses such as travel, parking, and child care, or providing extra incentives for completing follow-up. Researchers should be mindful, however, that one downside to some of these approaches is that they might influence participants' use of the study product or other behaviors related to the study outcome, which is detrimental if the goal of the trial is to estimate effectiveness during "typical use" of the product.In our study, participants who enrolled at study centers where enrollment was slow were at increased risk of failure to complete the study. The reason for this association is unclear. Factors at these centers that hindered enrollment also may have adversely affected participants' interest in remaining in the study. Alternatively, in responding to pressure to hasten recruitment, these centers may have enrolled women who were not good candidates for study completion. This latter possibility emphasizes the need to maintain a careful balance between recruitment and retention goals: rapid recruitment of participants who then drop out of the study is not beneficial to the study as a whole.The amount of reimbursement promised to our participants was strongly associated with final completion status in the bivariable analysis, but this effect was not significant when adjusted for other factors in our multivariable model. Numerous prior studies have shown that modest monetary incentives (e.g., $20 or less) increase response rates to surveys or short follow-up studies ,11 Some We were surprised that several of the factors that we expected would be associated with early withdrawal did not show significant associations in this analysis. When we began this analysis, we presumed that one reason for both slow enrollment and poor follow-up rates in barrier method trials is the relatively poor efficacy of these products: women may consider them to be temporary or backup methods and thus may be unwilling to use them as their sole or primary contraceptive for the 6–12 month duration of these studies. However, in our study, participants who strongly wished to avoid pregnancy or who had completed their desired family size were not more likely than others to drop out, nor were participants who expressed concerns about contraceptive efficacy early in the trial. Furthermore, neither early medical problems nor other complaints about the spermicides were predictive of withdrawal. These findings differ from that of a previous randomized trial of spermicides conducted mostly in developing countries. In that trial, participants who initially liked the assigned product very much were more likely than others subsequently to complete the study and to use the product for a longer period of time after admission .In one respect, the poor retention rate in our study and in other barrier method trials is a result of the design of these studies, which typically call for censoring data  when participants stop relying on the assigned contraceptive method. This design prohibits a true intent-to-treat analysis and is consequently a potential source of bias. Clearly, retention would be higher if the trials were designed at the outset to follow all subjects for the full intended duration of follow-up, even if they switched contraceptive methods. However, data from participants who are not using the method under study are not necessarily relevant to the efficacy and safety of the method. For the results of these trials to be meaningful, as many subjects as possible must not only complete follow-up but also continue to use the method during the full follow-up period. In our study, almost all the women who gave a reason for withdrawing early either cited problems with the spermicide or indicated that they wished to switch to another method after leaving the study. Our results are consistent with the findings of the 1995 National Survey of Family Growth, which showed that more than 47% of US spermicide users stopped relying on the method within the first 6 months of use . These fOur results suggest that to reduce bias potentially introduced by a large proportion of participants failing to complete the study, future barrier contraceptive method researchers should consider approaches in addition to those directly aimed at tracking and retaining individual participants. For example, both to reduce the burden on participants and to help the study staff maintain focus on follow-up, limiting data collection to critical variables may be appropriate. Complete collection of key data is clearly preferable to inadequate collection of less important data. Reducing the planned duration of follow-up would also certainly reduce withdrawals; although a larger sample size would be needed to provide the desired levels of precision and power, this disadvantage might be overcome if the shorter study were more attractive to potential participants. Given the large proportion of women who stop using the method earlier than 6 months, it is not clear that 6-month pregnancy probabilities are clinically needed anyway. Adding a run-in period to the trial before randomization might be helpful in excluding participants likely to drop out very early after admission, although such an addition might deter enrollment of other women as well, which is also a problem in these trials. Finally, innovative study designs to measure product efficacy should be evaluated. The design proposed by Steiner et al., which compares the one-month pregnancy probability in a relatively small number of women using a contraceptive method to the probability in women using a placebo, offers an alternative to the traditional 6–12 month trial . It showNo authors have any declared interests except the following:Elizabeth Raymond owns stock in Johnson and Johnson.Mitchell Creinin serves as a speaker for Ortho.Alfred Poindexter has had research grants from Columbia Laboratories and serves as speaker for Ortho.EGR helped design the trial, managed the trial, planned this analysis, and drafted the manuscript.PLC and BPL helped design the trial and/or this analysis, performed the analysis, and contributed to the manuscript.JL designed the trial and contributed to the manuscript.Other authors participated in the design of the trial, conducted the trial, and contributed to the manuscript.The pre-publication history for this paper can be accessed here:


S. cerevisiae, C. elegans, E. coli, A. thaliana, D. melanogaster, and H. sapiens. We use genomic sequence information to connect these data and compare global and modular properties of the transcription programs. Linking genes whose expression profiles are similar, we find that for all organisms the connectivity distribution follows a power-law, highly connected genes tend to be essential and conserved, and the expression program is highly modular. We reveal the modular structure by decomposing each set of expression data into coexpressed modules. Functionally related sets of genes are frequently coexpressed in multiple organisms. Yet their relative importance to the transcription program and their regulatory relationships vary among organisms. Our results demonstrate the potential of combining sequence and expression data for improving functional gene annotation and expanding our understanding of how gene expression and diversity evolved.Comparing genomic properties of different organisms is of fundamental importance in the study of biological and evolutionary principles. Although differences among organisms are often attributed to differential gene expression, genome-wide comparative analysis thus far has been based primarily on genomic sequence information. We present a comparative study of large datasets of expression profiles from six evolutionarily distant organisms:  Comparative analysis of sequence and gene expression data from bacteria, yeast, worms, flies, weeds, and humans hints at the potential to extract biological and evolutionary principles We performed a statistical analysis comparing the pairwise correlations between genes in one organism to the correlations between their respective homologues. Indeed, a significant fraction of such correlations were similar see . The strSaccharomyces cerevisiae expression data , while in the other organisms they may be required to accelerate folding during cell growth.The observation that groups of functionally related genes are often coexpressed in multiple organisms prompted us to ask whether also the higher-order regulatory relationships between these groups have been conserved . To addrC. elegans), the standard deviations of the correlation coefficients do not exceed 0.1, even when removing half of the expression profiles. Taken together, these results indicate that, despite the sparseness of the data, our findings reflect real properties of the expression networks and not the specific subset of experimental conditions used.In order to test whether the variations in the regulatory relations among functional groups in different organisms are due to the use of unrelated sets of experimental conditions, we restricted both the human and the yeast expression data to the cell cycle experiments. We found that the correlations between modules did not change qualitatively due to this restriction B and 2C.To compare the higher-order regulatory structures more systematically, we decomposed the expression data of each organism into a set of transcription modules using the iterative signature algorithm (ISA) we proposed recently A and 3B.C. elegans tree exhibits a sharp transition between a regime dominated by a single branch (from which only few less-stable modules branch off) to a part of the tree that rapidly bifurcates into many branches at higher thresholds , most modules have either significantly less or significantly more homologues than expected  of the connectivity k (the number of edges of a particular gene). We find that for all organisms, the connectivity is distributed as a power-law, n(k) ∼ k−γ, with similar exponents γ ≈ 1.1–1.8  that two genes of connectivity k and k′, respectively, are connected with each other . Interestingly, in the three organisms in which large-scale knockout information is available  identified by our modular analysis. The decrease of  modules E. Thus, Comparing genomic properties of different organisms is of fundamental importance in the study of biological and evolutionary principles. Although much of the differences among organisms is attributed to different gene expression, comparative analysis thus far has been based primarily on genomic sequence information. The potential of including functional genomic properties in a comparison analysis was demonstrated in recent studies that compared protein–protein interaction networks of different organisms .In this paper we presented a comparative analysis of large datasets of expression profiles from six evolutionarily distant organisms. We showed that all expression networks share common topological properties, such as a scale-free connectivity distribution and a high degree of modularity. While these common global properties may reflect universal principles underlying the evolution or robustness of these networks, they do not imply similarity in the details of the regulatory programs. Rather, with a few exceptions, the modular components of each transcription program as well as their higher-order organization appear to vary significantly between organisms and are likely to reflect organism-specific requirements.http://barkai-serv.weizmann.ac.il/ComparativeAnalysis/.Nevertheless, coexpression of functionally linked genes is often conserved among several organisms. Based on this finding, we proposed an efficient method that uses coexpression analysis for improving sequence-based functional annotation. An interactive implementation of this algorithm is available at Our analysis was based on the available expression data, which are still sparse for most organisms. It is likely that the modular decompositions we obtained are partial, so additional modules can be identified as more expression data become available. Nevertheless, by analyzing the sensitivity of our results to the number of conditions, we concluded that the composition of the modules themselves is rather robust. Moreover, the higher-order correlations between modules are only slightly affected by the number of conditions.S. cerevisiae . We excluded genes or conditions with more than 90% missing datapoints, resulting in expression matrices of the dimensions shown in Preprocessed expression data from Database  using deDatabase , we onlyFASTA files for amino-acid sequences of coding regions were downloaded from the sources detailed in S. cerevisiae and E. coli) and RNAi experiments (C. elegans) were obtained from the sources indicated in Data for deletion mutants (mG of the genome G) and an associated set of regulating conditions . The defining property of a transcriptional module is self-consistency, which is achieved as follows. First, we assign scores to both genes and conditions that reflect their degree of association with the module. The gene score is the average expression of each gene over the module conditions, weighted by the condition score: g in condition c normalized over genes and conditions, respectively, such that gs, while the module conditions are those conditions in the dataset with the highest scores cs. The ISA identifies transcription modules through iterative refinement of a large number of random gene scores.A transcription modules consist of a set of coregulated genes  in the present analysis, but retained only genes whose score is not less than 70% of the most significant gene  is identical in all expression networks and fixed it to <k> = 0.001. Using the top 0.1% of all possible correlations corresponds to a lower limit on the correlation coefficients between 0.63 for S. cerevisae and 0.85 for D. melanogaster. The results are insensitive to the precise threshold value (see i is k = Σj≠iijA. In order to obtain the connectivity distributions n(k), we used logarithmic binning. The edges of the bins were powers of 2, and we counted the number of genes with ik between two edges and normalized by the bin width. We applied a linear fit to the log values of the bin centers against the normalized counts. We note that the resulting connectivity distributions scale as a power-law for a wide range of thresholds and the exponents only depend weakly on the choice of the threshold. The clustering coefficient of gene i is iC = (Σk>j≠iikAkjAjiA)/[ik(ik −1)/2].Each expression network can be described by a symmetric adjacency matrix http://barkai-serv.weizmann.ac.il/ComparativeAnalysis.Interactive applications for the refinement of sets of homologous genes and the exploration of our modular decompositions of the expression data are available online. We also present details about the highly connected genes in each organism, the pairs of genes that are significantly correlated in two organisms, and the eight modules related to core processes in yeast (and their homologue modules before and after refinement) on our website at Data S1Figure S1 and Figure S2.This note includes (38 KB PDF).Click here for additional data file.Data S2Figure S3, Figure S4, and Figure S5.This note includes (59 KB PDF).Click here for additional data file.Data S3Figure S6.This note includes (11 KB PDF).Click here for additional data file.Data S4(3 KB PDF).Click here for additional data file.Data S5After this work was completed, we succeeded in processing the more than 2,000 human chip experiments deposited at the SMD. Removing genes and conditions with more than 90% missing values resulted in 1,474 expression profiles for 24,795 genes. Our Web tools (“GeneHopping” and “ModuleTree”) allow researchers to use also this updated dataset.(3 KB PDF).Click here for additional data file.Figure S7(137 KB PDF).Click here for additional data file.Figure S8(16 KB PDF).Click here for additional data file.Figure S9(15 KB PDF).Click here for additional data file.Figure S10(33 KB PDF).Click here for additional data file.Figure S11(11 KB PDF).Click here for additional data file.Figure S12(29 KB PDF).Click here for additional data file.Figure S13(19 KB PDF).Click here for additional data file.


Inconsistent findings have been reported on the occurrence and relevance of creatine kinase (CK) isoenzymes in mammalian liver cells. Part of this confusion might be due to induction of CK expression during metabolic and energetic stress.The specific activities and isoenzyme patterns of CK and adenylate kinase (AdK) were analysed in pathological liver tissue of patients undergoing orthotopic liver transplantation.The brain-type, cytosolic BB-CK isoenzyme was detected in all liver specimens analysed. Conversely, CK activity was strongly increased and a mitochondrial CK (Mi-CK) isoenzyme was detected only in tissue samples of two primary hepatocellular carcinomas (HCCs).The findings do not support significant expression of CK in normal liver and most liver pathologies. Instead, many of the previous misconceptions in this field can be explained by interference from AdK isoenzymes. Moreover, the data suggest a possible interplay between p53 mutations, HCC, CK expression, and the growth-inhibitory effects of cyclocreatine in HCC. These results, if confirmed, could provide important hints at improved therapies and cures for HCC. Creatine kinase (CK) isoenzymes catalyse the reversible transfer of the phosphate group of phosphocreatine (PCr) to ADP, to yield ATP and creatine (Cr). The CK/PCr/Cr system is present primarily in tissues with high and fluctuating energy demands such as brain, heart and skeletal muscle, and serves as a temporal and spatial "energy buffer" that helps to maintain a high intracellular phosphorylation potential in situations of increased metabolic demand (for reviews, see ,2).de novo. The liver is the main site of Cr production in the body . After ody see -6). Othe. Othede ody . Finallody  isoenzyme. On the other hand, besides BB-CK which was suggested to be present in endothelial and Kupffer cells, Vaubourdolle et al.  also proIn a number of studies reporting significant levels of CK activity in liver, interference by adenylate kinase (AdK) isoenzymes in the CK activity assays -21 is veBecause of these conflicting data, the goal of the present study was to analyse in detail the CK and AdK activities in pathological liver tissue of patients undergoing orthotopic liver transplantation.The present project was approved by the ethics commission of the University of Innsbruck. In total, 25 liver samples were analysed. Twenty-three samples were obtained from 18 explanted organs of liver transplant recipients, one sample was obtained at autopsy (no. 1), and the last sample was from a normal rat liver. According to pathomorphological criteria, the 25 samples can be divided into 5 groups: (1) Nine samples of cirrhotic liver tissue ; (2) six samples of neoplastic tissue ; (3) three samples of necrotizing liver tissue due to acute or subacute organ rejection (nos. 2, 6, 22); (4) five samples of macroscopically normal liver parenchymal tissue (nos. 3, 8, 10, 16, 25) surrounding focal liver pathologies ; (5) two samples originating from a normal rat liver (no. 12) and from a patient with steatosis hepatis (no. 1).All steps were performed on ice or at 4°C. Approximately 5 g of liver tissue was homogenized in 45 ml buffer A (250 mM sucrose, 5 mM HEPES, 0.5 mM EGTA, pH 7.4). The homogenate was subjected to centrifugation for 5 min at 800 g. The pellet was discarded, and the supernatant centrifuged for 4 min at 5,100 g (centrifugation C2). The supernatant of C2 was further clarified by centrifugation for 12 min at 12,300 g, thus yielding the cytosolic fraction. The pellet of C2 was resuspended in 10 ml buffer A, followed by centrifugation for 2 min at 12,300 g (C3). After resuspension of the C3 pellet in 10 ml buffer A and centrifugation for a further 10 min at 12,300 g, the sediment was resuspended in 4 ml buffer A, thus yielding the mitochondrial fraction. One-ml aliquots of the different fractions were immediately frozen in liquid nitrogen and stored at -80°C until analysis.2, 2.1 mM ADP, 2.1 mM NADP, 21 mM N-acetylcysteine, 9 U/ml of hexokinase, and 5.8 U/ml of glucose-6-phosphate dehydrogenase (both from Sigma). Enzymatic activity was measured at 25°C as an increase in NADPH absorbance at 340 nm. For AdK, three separate measurements were made for each sample in the same assay medium. For CK measurements, 5.1 mM AMP was added to the assay medium to inhibit AdK activity. For each sample, three measurements with 10.3 mM PCr and three measurements without PCr  were made, and the CK activity was calculated as the difference of the respective means. All values in this paper represent specific activities per mg of homogenate, cytosolic or mitochondrial protein. Protein amounts were measured according to the method of Bradford [For CK and AdK activity measurements, the following assay medium was used: 110 mM imidazole, pH 6.7, 20.5 mM glucose, 11 mM Mg-acetate, 2.05 mM EDTA·NaBradford  with bovCPE was performed at room temperature for 90 min at a constant voltage of 150 V, but otherwise as described previously . CK and -1, respectively. In most other liver samples, the specific CK activities in the homogenate were below 0.05 U·(mg protein)-1. Whereas enzymatic activity measurements revealed low, but consistent CK activity in many of the cytosolic fractions, no CK activity was detected in the mitochondrial fractions, except for HCC sample no. 9 with a specific CK activity of approx. 0.1 U·(mg protein)-1 (due to limited sample size, subcellular fractionation was not feasible for HCC sample no. 4). These findings were corroborated qualitatively by isoenzyme electrophoresis on cellulose polyacetate strips. Visualization of the different CK isoenzymes by an overlay gel technique revealed that the brain-type cytosolic BB-CK isoenzyme was present in all liver samples. Conversely, bands for the dimeric and octameric forms of Mi-CK were only observed in the two primary HCC samples , 0.5 for liver sample no. 5 (secondary biliary cirrhosis), and < 0.2 for all other liver samples. Similar findings were made for the cytosolic and mitochondrial fractions, with CK/AdK activity ratios of < 0.3 for the cytosolic fraction and < 0.05 for the mitochondrial fraction. For HCC sample no. 9, however, these ratios were significantly higher: 4.8 (cytosolic fraction) and 0.33 .-1), levels which are comparable with or lower than data reported for rat and human liver [-1; [CK is an enzyme still widely analysed in clinical diagnostics. Although a wealth of CK measurements have been reported in the scientific literature, there still exist inconsistency and incomplete knowledge on such an apparently simple question as the CK (isoenzyme) content of mammalian liver in both health and disease. In the present study, we detected the presence of BB-CK in all liver samples analysed by using CK activity measurements and cellulose polyacetate electrophoresis. However, in the normal and most pathological liver samples that we analysed, the specific CK activity was very low . ver [-1; .in vitro of 1.4–2.6, the specific CK activities were still relatively low (0.21–0.36 U·[mg protein]-1). Therefore, in the absence of histochemical data, it cannot be concluded with certainty whether the increased levels of CK are due to increased vascularization of the tumour , or to induction of CK expression in the malignant cells.Interestingly, we observed a strong induction of both BB-CK and Mi-CK expression in two samples of primary HCC. Despite CK/AdK activity ratios Induction of CK expression has been observed previously in many types of tumours (see ) and mayA key player in the picture might be the p53 tumour suppressor gene. Mutations in p53 are quite prevalent in HCC, especially in tumours with low cellular differentiation ,43. On tin vivo models  the limited responsiveness of HCC to currently available therapeutic approaches and, thus, (ii) the poor prognosis associated with this disease. Cr analogues (cyclocreatine and β-guanidinopropionic acid) and also Cr itself were previously shown to have antitumour activity, both in cell culture and in  models . Similaepatoma; .The present findings shed light on some old enigmas and open up fascinating avenues for future research. Our findings do not support significant expression of CK in normal liver and most liver pathologies, but rather indicate that many of the previous misconceptions in this field can be explained by interference from AdK isoenzymes. On the other hand, given the need for improved understanding of the molecular pathogenesis of HCC, and for improved therapies and cures, the induction of CK expression in HCC described here calls for a more in-depth analysis of the interplay between p53 mutations, HCC, CK expression, and the growth-inhibitory effects of cyclocreatine in HCC.AdK, adenylate kinase; BB-CK, brain-type cytosolic CK isoenzyme; CK, creatine kinase; Cr, creatine; HCC, hepatocellular carcinoma; Mi-CK, mitochondrial CK; MM-CK, muscle-type cytosolic CK isoenzyme; PCr, phosphocreatine.The authors declare that they have no competing interests.GM and RM covered the medical part of this study. GM, FNG and MW performed the biochemical experiments. MW drafted the manuscript.The pre-publication history for this paper can be accessed here:


Caenorhabditis elegans vulval development, the anchor cell (AC) in the somatic gonad secretes an epidermal growth factor (EGF) to activate the EGF receptor (EGFR) signaling pathway in the adjacent vulval precursor cells (VPCs). The inductive AC signal specifies the vulval fates of the three proximal VPCs P5.p, P6.p, and P7.p. The C. elegans Rhomboid homolog ROM-1 increases the range of EGF, allowing the inductive signal to reach the distal VPCs P3.p, P4.p and P8.p, which are further away from the AC. Surprisingly, ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. This observation led to the discovery of an AC–independent activity of EGF in the VPCs that promotes vulval cell fate specification and depends on ROM-1. Of the two previously reported EGF splice variants, the longer one requires ROM-1 for its activity, while the shorter form acts independently of ROM-1. We present a model in which ROM-1 relays the inductive AC signal from the proximal to the distal VPCs by allowing the secretion of the LIN-3L splice variant. These results indicate that, in spite of their structural diversity, Rhomboid proteins play a conserved role in activating EGFR signaling in C. elegans, Drosophila, and possibly also in mammals.During  ROM-1 increases the range of EGF, which specifies the vulval fate of precursor cells, by functioning in the signal- receiving precursor cells rather than the signal-sending anchor cells Intercellular signaling pathways control many diverse processes, such as cell proliferation, differentiation, survival, migration, shape changes, and responses to the environment. In most instances, the release of the signaling molecules by the signal-sending cells constitutes a rate-limiting step that determines the spatial distribution and temporal duration of the response . On the Drosophila growth factor Spitz, which activates the EGFR in multiple developmental processes  receptor (EGFR) acts in a highly conserved signal transduction pathway that controls various cell fate decisions in metazoans . EGFR lirocesses . Geneticing cell . Drosophroteases . Site-sproteases . The Dro pathway . There a elegans . Rhomboi elegans . Rhomboi elegans  and the ease S2P . On the ease S2P . WhetherC. elegans hermaphrodite vulva serves as a simple model by which to study signal transduction and cell fate determination during organogenesis (C. elegans postembryonic development, the anchor cell (AC) in the somatic gonad induces three out of six equivalent vulval precursor cells (the VPCs, termed P3.p through P8.p) in the ventral hypodermis to adopt vulval cell fates , rom-2 (C48B4.2), rom-3 (Y116A8C.14), rom-4 (Y116A8C.16), and rom-5 (Y54E10A.14) , followed by ROM-2 (29% identity) and the more diverged ROM-3 (24% identity), ROM-4 (26% identity), and ROM-5 (29% identity). Mutagenesis experiments with Drosophila Rhomboid-1 have identified a catalytic triad formed by conserved asparagine, serine, and histidine residues that are necessary for the serine protease activity (.Since the LIN-3 EGF growth factor is produced as a transmembrane precursor protein , we askeE10A.14) A. All fihomboids . The trahomboids B. ROM-1 activity . This caactivity B, suggesrom-1, we isolated rom-1 cDNA by RT-PCR. An SL1 trans-spliced leader sequence was identified at the 5′ end of the message that was spliced to the second of the six exons predicted by the C. elegans genome project (http://www.wormbase.org). The remaining intron-exon boundaries were confirmed experimentally and corresponded exactly to the predicted boundaries. The conceptual translation of the 1,071-bp open reading frame (ORF) predicts a protein of 356 amino acids, with very short stretches of hydrophilic amino acids between the seven-pass transmembrane domains, except for a longer loop consisting of 43 amino acids between the first and second transmembrane domains (see In order to confirm the predicted intron-exon structure of  project C (see htains see B.rom genes, we used RNA interference (RNAi) to transiently knock down their expression  animals with bacteria producing rom-3 dsRNA had no effect on vulval development (unpublished data). Due to the high degree of sequence similarity between rom-3 and rom-4 (69.8% identity), rom-3 RNAi is likely to simultaneously reduce rom-4 function.As a first step to examine the biological function of the pression . Double-rom-1 gene (see zh18 deletion removes 206 amino acids from the N terminus, including the first three transmembrane domains and 384 bp of promoter sequences. Thus, the zh18 deletion probably results in a complete loss of rom-1 function and will be referred to as rom-1(0). The rom-1(0) single mutants exhibited no obvious phenotype; they were healthy and fertile. In addition, we obtained the rom-2(ok966) allele from the C. elegans Gene Knockout Consortium. The rom-2(ok966) animals carry a 530-bp deletion that removes the fifth exon, which contains the predicted catalytic center with the essential histidine residue (see rom-2(rf). Consistent with the RNAi experiments, both rom-1(0) and rom-2(rf) single mutants exhibited normal vulval development (rom-1(0) rom-2(rf) double mutants, no defects in vulval development were observed, ruling out a possible redundant function of the two genes  mutation as well as rom-1 RNAi partially suppressed the multivulva (Muv) phenotype caused by overexpression of the LIN-3 EGF growth factor [lin-3(+)] (n1046 gain-of-function (gf) mutation in the let-60 ras gene, which renders vulval development partially independent of upstream signaling (rom-1(0) mutation suppressed the Muv phenotype of hs::mpk-1 animals that overexpress the wild-type MAPK MPK-1 under control of a heat-shock promoter together with Drosophila MEK-2 under control of the interferon-1α promoter (rom-1, neither the rom-2(rf) mutation nor rom-2 RNAi affected the Muv phenotype of let-60(gf) animals (Next, we examined whether loss of in-3(+)]  or by thignaling  (Table 1promoter  (Table 1rom-1(0) mutation did not significantly enhance the vulvaless (Vul) phenotype caused by the lin-3(e1417), lin-2(n397), sem-5(n2019), or let-60(n2021) mutations that reduce the activity of the receptor tyrosine kinase (RTK)/RAS/MAPK pathway (unpublished data). Since these Vul mutants affect the cell fates of only the proximal VPCs (P5.p, P6.p, and P7.p), ROM-1 plays no role in the induction of the proximal VPCs by the AC. Thus, ROM-1 enhances the activity of the EGFR/RAS/MAPK pathway to allow the induction of the distal VPCs P3.p, P4.p, and P8.p.The (hs::lin-3extra) (rom-1(0) (lin-15(rf) mutants, all VPCs adopt vulval cell fates independently of the LIN-3 signal, though induction in lin-15(rf) mutants depends on the activity of LET-23 and the other components of the EGFR/RAS/MAPK pathway (rom-1(0) mutation did not suppress the Muv phenotype of lin-15(rf) animals, suggesting that loss of rom-1 function affects the LIN-3-dependent induction of vulval cell fates rather than the LIN-3-independent activity of the EGFR/RAS/MAPK pathway  . Unlike  pathway . The romrom-1 and the Notch and Wnt pathways, since both pathways control vulval cell fate specification in parallel with the RTK/RAS/MAPK pathway (lin-12 notch(gf) animals, no AC is formed, and all VPCs adopt the 2° cell fate (rom-1(0) lin-12(gf) double mutants  mutation  mutation on the egl-17::cfp expression pattern in mid L2 larvae. For this purpose, larvae were synchronized in the mid L1 stage at 13 h of development by letting them hatch in the absence of food, and then development was allowed to proceed by adding food for another 24 h until they reached the mid L2 stage (approximately 37 h of development). Loss of ROM-1 function had no effect on egl-17::cfp expression in the proximal VPCs (P5.p, P6.p, and P7.p), but significantly reduced egl-17::cfp expression in the distal VPCs when compared to wild-type animals (egl-17::cfp expression pattern in rom-1(0) animals is consistent with the epistasis data, which showed that loss of rom-1 function affects the induction of only the distal VPCs (see above).To assess how much inductive signal each VPC receives, we examined the expression pattern of the  pathway . In mid and P7.p , and lowand P7.p A and 2B. animals C and 2D.rom-1 reporter by fusing 6.9 kb of the 5′ rom-1 promoter/enhancer region to the green fluorescent protein (gfp) ORF carrying a nuclear localizing signal (zhIs5[rom-1::nls::gfp]). With a translational full-length rom-1::gfp fusion construct, we failed to obtain transgenic lines that consistently expressed ROM-1::GFP. Moreover, a genomic DNA fragment encompassing the entire rom-1 locus failed to produce stable transgenic lines even when injected at relatively low concentrations (1–10 ng/μl), suggesting that elevated levels of ROM-1 are toxic to the animals.To analyze the expression pattern of ROM-1, we generated a transcriptional rom-1::nls::gfp reporter was widely expressed in somatic cells throughout development. Surprisingly, we did not detect any rom-1::nls::gfp expression in the gonadal AC before the L4 stage, while consistent expression was observed in the Pn.p cells and the Pn.a-derived neurons from the L1 stage on. In early L2 zhIs5 larvae, the six VPCs expressed rom-1::nls::gfp at equal levels . After vulval induction, rom-1::nls::gfp was down-regulated in the 1° and 2° descendants of P5.p, P6.p and P7.p, while the 3° descendants of P3.p, P4.p, and P8.p again expressed high levels of rom-1::nls::gfp after they had fused with hyp7 . To test whether rom-1::nls::gfp expression depends on RTK/RAS/MAPK signaling in the VPCs, we introduced the zhIs5 transgene into lin-7(e1413) mutants that exhibit a penetrant Vul phenotype due to reduced LET-23 EGFR activity (lin-7(e1413); zhIs5 animals, the up-regulation of rom-1::nls::gfp occurred less frequently (in 13%, 33%, and 7% of the cases in P5.p, P6.p, and P7.p, respectively, n = 15). Thus, the AC signal up-regulates rom-1::nls::gfp expression in the VPCs that adopt vulval cell fates.The transcriptional l levels A and 3B.al fates C and 3D.ith hyp7 E and 3F.r fusion G and 3H r fusion . To test1 and Z4 . Uniformactivity . In lin-rom-1(+) function on vulval induction in gonad-ablated animals. If ROM-1 acts exclusively in the AC, then the rom-1(0) mutation should not affect vulval induction in gonad-ablated animals. On the other hand, if ROM-1 acts in cells other than the AC, then the rom-1(0) mutation should suppress vulval induction even in the absence of the AC. Since the inductive AC signal is absolutely required to initiate vulval development (let-60(gf) or hs::mpk-1 animals that exhibit a hyperactive EGFR/RAS/MAPK signaling pathway causing AC-independent vulval induction (lin-3(+) animals because, as reported previously by lin-3 under control of its own promoter, some vulval differentiation could still be observed in the absence of the AC, pointing at an additional source of LIN-3 from the transgene in cells outside of the gonad (rom-1 function in gonad-ablated lin-3(+), let-60(gf), or hs::mpk-1 animals caused a strong further reduction in vulval induction (lin-15(rf) animals that exhibit lin-3 independent vulval differentiation was not affected by the rom-1(0) mutation (Table2, rows 13 and 14).To examine whether ROM-1 acts in cells other than the AC (which is part of the somatic gonad), we tested the effect of loss of elopment , we perfnduction . In addihe gonad , row 2. egl-17::cfp expression pattern after removal of the AC. In gonad-ablated animals, residual egl-17::cfp expression was observed in all VPCs  loss-of-function mutation [lin-3(0)] was much stronger than the decrease observed in gonad-ablated lin-3(+); hs::mpk-1 animals (lin-3(0) mutation was suppressed by the hs::mpk-1 transgene). Vulval induction in lin-3(0); hs::mpk-1 animals was not affected by gonad ablation since the lin-3(0) allele eliminated lin-3 function in the AC  reduction-of-function mutation almost completely abolished the expression of the egl-17::cfp marker (egl-17::cfp expression in the VPCs. Loss of rom-1 function in a lin-3(0); hs::mpk-1 background caused no further decrease in vulval induction, suggesting that ROM-1 does not affect vulval development in the absence of LIN-3  ; let-60(gf) and rom-1(0); hs::mpk-1 double mutants to levels comparable to those found in let-60(gf) and hs::mpk-1 single mutants (lin-31 promoter (lin-31::cre) that was used as a negative control had no effect on vulval induction ; let-60(gf) animals (rom-1 in the AC under control of the AC-specific enhancer (ACEL) (ACEL::rom-1), which is located in the third intron of the lin-3 locus (lin-31::rom-1, the ACEL::rom-1 transgene did not rescue the suppression of the let-60(gf) Muv phenotype by rom-1(0) (rom-1 function.The absence of detectable ::rom-1) . The linnduction . Consist-3 locus . In contlin-3 dsRNA in the Pn.p cells in order to down-regulate by RNAi any possible lin-3 expression in the VPCs (lin-3 cDNA fragment under control of the same Pn.p cell-specific lin-31 promoter used above (lin-31::lin-3i) was introduced into wild-type animals . In wild-type L4 larvae, the 1° descendants of P6.p in the vulF toroid ring , secrete LIN-3 to specify the ventral uterine (uv1) cell fate in the somatic gonad . The lin-31::lin-3i transgene almost completely suppressed the Muv phenotype caused by the lin-31::lin-3S transgene, while the lin-31::cre transgene that was used as negative control had no effect  and hs::mpk-1 animals  animals lacking a gonad  single mutants exhibited wild-type levels of vulval induction, the egl-38(rf) mutation reduced the Muv phenotype of hs::mpk-1 animals to a similar degree as the rom-1(0) mutation or the lin-31::lin-3i transgene  produce LIN-3 to amplify the inductive signal.lin-3 locus encodes two splice variants termed LIN-3S (short) and LIN-3L (long) that are generated by the differential choice of the splice donor of exon 6  transgenic lines generated by injection of a relatively low (1 ng/μl) or high (100 ng/μl) concentration of the lin-3S minigene exhibited a strong Muv phenotype (with induction indices ranging from 4.1 to 5.6). In contrast, the lin-3L construct caused a Muv phenotype only when injected at a high concentration. . For the lin-3XL construct, we obtained variable results; some lines exhibited a weak Muv and others no or even a Vul phenotype (unpublished data). Since we failed to observe a consistent phenotype with this minigene construct, we did not further pursue the analysis of the lin-3XL minigene.The f exon 6 B  background. The lin-3S and lin-3L transgenes both rescued the larval lethality of lin-3(0) mutants, yielding adult Muv animals (lin-3(0); lin-3S and lin-3(0); lin-3L animals were sterile. Loss of rom-1 function did not affect the viability or the Muv phenotype of lin-3(0); lin-3S animals  mutants was reduced by loss of rom-1 function (rom-1(0), lin-3(0); lin-3L animals that escaped the larval lethality exhibited a weaker Muv phenotype than lin-3(0); lin-3L animals, suggesting that the function of LIN-3L during vulval induction partially depends on ROM-1 activity . The lin-31::lin-3S and lin-31::lin-3L transgenes both caused a strong Muv phenotype in the presence and absence of the AC  mutants an unprocessed, membrane-bound form of LIN-3 that is retained on the plasma membrane of the AC induces the 1° fate in the adjacent VPC P6.p through juxtacrine signaling . It is possible that the reporter constructs used were lacking some of the regulatory sequences necessary to drive strong lin-3 expression in the VPC lineage. Other potential sources of LIN-3 may be the posterior ectoderm or the excretory system in the head. However, it seems unlikely that LIN-3 secreted from cells at the anterior or posterior end of the animal influences vulval induction, since we did not observe a bias favoring the induction of anterior or posterior VPCs in the absence of the AC.Three lines of evidence indicate that ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. First, a omboid-1  suggest he gonad  and by tal cells , had essL4 stage , and occrom-1::nls::gfp are highest in the proximal VPCs (P5.p, P6.p, and P7.p) that adopt 1° and 2° vulval cell fates, suggesting that the proximal VPCs are competent to secrete LIN-3 in response to the inductive AC signal. LIN-3 from the proximal VPCs may facilitate the induction of the more distally located VPCs by paracrine signaling  mutation, including the ovulation defects (unpublished data). This may explain why loss of rom-1 function causes neither the larval lethality nor the sterility observed in lin-3 mutants. Furthermore, our data indicate that LIN-3L function in the VPCs almost completely depends on ROM-1 activity. It seems improbable that the 15 amino acid insertion in LIN-3L could change the substrate specificity toward the ROM-1 protease. A more likely explanation for the inherent difference in the dependence of the LIN-3 splice variants on ROM-1 is suggested by the experiments performed with Drosophila Spitz  as well as the newly identified longer variant (LIN-3XL) differ by 15 and 41 amino acid insertions in the juxtamembrane region just prior to the predicted Rhomboid cleavage site at the start of the transmembrane domain . Our expla Spitz . When exla Spitz , tissue-tebrates . Differetebrates . The tisCaenorhabditis elegans  (dpy-19(e1259), lin-12(n137gf), rom-1(zh18) (this study), rom-2(ok966) (C. elegans Gene Knockout Consortium), and unc-119(e2498); LGIV: let-60(n1046) (lin-3(n1049), unc-5(e53), unc-44(362), lin-45(sy96), unc-24(e138), mec-3(e1338), dpy-20(e1282), egl-38(n578), and mec-3(n3197); LGX: sem-5(n2019) and lin-15(n309); extrachromosomal and integrated arrays: zhEx22[lin-3(+), sur-5::gfp, unc-119(+)], zhE66[lin-31::rom-1, unc-119(+), sur-5::gfp], zhEx72[lin-31::lin-3S, unc-119(+), sur-5::gfp], zhEx68[lin-3S, unc-119(+), sur-5::gfp], zhEx69[lin-3L, sur-5::gfp, unc-119(+)],zhEx73[lin-31::lin- zhEx73[lin-31::lin-3L, unc-119(+) sur-5::gfp], zhEx78[ACEL::Δpes-10::nls::gfp, unc-119(+)], zhEx81[lin-31::cre, unc-119(+), myo-3::gfp], zhE88[lin-31::lin-3i, unc-119(+), myo-3::gfp], zhEx89[ACEL::rom-1, sur-5::gfp], syIs12[hs::lin-3extra] (zhIs5[rom-1::nls::gfp, unc-119(+)], huIs7[hs::bar-1ΔNT, dpy-20(+)] , and pTG96 (sur-5::gfp, 100 ng/μl) were used as a cotransformation markers ] was integrated in the genome animals following γ-irradiation with 3,000 Rad to generate the array zhIs5 and backcrossed six times before analysis. Double and triple mutants were constructed using standard genetic methods. Where cis-linked markers were used they are indicated in the table legends.Standard methods were used for maintaining and manipulating  elegans . The C. -1(mu38) ; LGIII: 0(n1046) , lin-3(n-3extra] , zhIs5[ry-20(+)] , gaIs36[-Dmek-2] , and arI17::cfp] . Unless 17::cfp] .The cons markers . The extrom-1::nls::gfp reporter construct (pRH2) was generated by ligating a HindIII-NheI–restricted 6,998-bp genomic fragment spanning the entire 5′ upstream region of F26F4.3 isolated by PCR amplification with the primers OAD49 (5′-GGAAGCTTGCATGCCCAACGAAATCGATA-3′) and OAD59 (5′-GGGCTAGCCATGTTGTGGAGAAGGAGAAC-3′) into the HindIII-XbaI site of pPD96.04. The rom-1::gfp translational reporter construct (pAD31) was generated by PCR amplification of a 3,146-bp genomic fragment containing 1,849 bp of 5′ sequences and the entire rom-1 ORF using the primers OAD47 (5′-GACTCTAGAGTTGTCAAAAGGTCACGGG-3′) and OAD51 (5′-ATCCTCTAGAGTTGAGCAATTTTCGTTGTTCCAC-3′') followed by XbaI restriction and ligation to XbaI-digested vector pPD95.75. The upstream promoter region of this construct was further extended by replacing a 420-bp PstI fragment with a 2,099-bp PstI genomic fragment corresponding to positions –1,432 and –3,531 relative to the predicted translation start codon of F26F4.3. The lin-31::rom-1 construct (pAD16) was generated by ligating a 1,601-bp SalI-NotI fragment spanning the entire rom-1 coding sequence amplified with the primers OAD44 (5′-TTTTGGTCGACCTCCTTCTCCACAAC-3′) and OAD45 (5′-TTTGGCGGCCGCCTATGAGCAATTTTCG-3′) into the SalI-NotI site of the pB253 vector  showed strong and specific GFP expression in the AC beginning in the mid L2 stage as reported  and OAD32 (5′-CGTATCTGCAGAATCCAACTCGATATTAATTAC-3′) using first-strand cDNA synthesized from mixed-stage total RNA as template. The PCR-amplified products were size-fractionated by agarose gel electrophoresis, cloned into the pGEMT vector (Promega), and sequenced to identify clones encoding individual splice variants. To obtain full-length lin-3 cDNA construct (pAD27), a 1,996-bp XhoI fragment from the EST clone yk1053b07 (confirmed to encode full-length lin-3XL cDNA by DNA sequencing) was first subcloned into the XhoI site of a modified pBluescriptSK  vector (pAD23) in which the PstI site had been destroyed by restriction with EcoRV and SmaI and religation of the resulting blunt ends. To generate full-length lin-3S and lin-3L cDNA constructs (pAD25 and pAD26, respectively), 1,065-bp and 1,110-bp PstI fragments specific for each splice variant isolated from the partial cDNA clones described above were used to replace the 1,188-bp PstI fragment in the full-length lin-3XL cDNA construct (pAD27). The lin-31::lin-3S and lin-31::lin-3L constructs were generated by cloning the 1,133-bp and 1,178-bp XhoI cDNA fragments of the S and L splice variants into the SalI site of pB253 (lin-3 splice variant minigene constructs (pAH63, pAH64, and pAH65 for lin-3S, lin-3L, and lin-3XL, respectively) were generated by cloning a 6.1-kb genomic fragment spanning the entire ORF of lin-3 and 574 bp of 5′ and 236 bp of 3′ sequences amplified with the primers OAH137 (5′-CCAGAAAGTTCATGTGAATCAT-3′) and OAH138 (5′-TCACAGGAACTGAGAGGGAGAGTG-3′) into the pGEMT vector. From this construct, a 6,206-bp ApaI-SacI fragment was subcloned into pAD23 to obtain pAH62. The minigenes encoding each of the splice variants were obtained by replacing the 2,728-bp lin-3 genomic PstI fragment with 1,065-, 1,110-, and 1,188-bp PstI fragments isolated from cDNAs of the different splice variants. To construct the lin-31::lin-3 hairpin plasmid (pAD35), a 964-bp NdeI-HindIII lin-3S cDNA fragment from pAD25 was cloned into NdeI-HindIII–digested pAD27 using the recA–E. coli SURE strain as host to obtain pAD32. The resulting 1,918-bp lin-3 hairpin fragment was excised with XhoI from pAD32 and subcloned in E. coli SURE into the SalI site of the pB253 vector to obtain pAD35.The transcriptional 3 vector . To genethe ACEL , was clopromoter . Transgereported . The Kpnof pB253 . The linrom-1 function was generated by in vitro transcription using a 350-bp rom-1 cDNA fragment corresponding to the nucleotides –17 to 725 relative to the predicted start codon of the ORF inserted into pGEM-T (Stratagene) as template. Transcripts were prepared using T7 and Sp6 RNA polymerase and annealed prior to injection as described  deletion mutant was isolated from an ethyl methanesulfonate–mutagenized library consisting of approximately 106 haploid genomes as previously described (zh18 deletion removes 1,556 bp including 384 bp of 5′ upstream sequences and 618 bp of the rom-1 ORF (positions 4906804–4908360 in the cosmid F26F4). The mutant strain was backcrossed six times against N2 before further experiments were done.The escribed . DNA pooVulval induction was scored by examining worms at the L4 stage under Nomarski optics as described . The numhttp://www.ncbi.nlm.nih.gov/) accession numbers of the Rhomboid genes discussed in this paper are C.e. ROM-1 (AAA91218), C.e. ROM-2 (CAA82377), C.e. ROM-3 (CAB55154), C.e. ROM-4 (CAB55122), C.e. ROM-5 (AAF60768), D.m. Rho-1 (CAA36692), D.m. Rho-2 (AAK06752), D.m. Rho-3 (AAK06753), D.m. Rho-4 (AAK06754), D.m. Rho-6 (NP_523557), H.s. Rho-1 (CAA76629).The GenBank (