Interspecies and in vitro-in vivo scaling for quantitative modeling of whole-body drug pharmacokinetics in patients: Application to the anticancer drug oxaliplatin.

Quantitative systems pharmacology holds the promises of integrating results from laboratory animals or in vitro human systems into the design of human pharmacokinetic/pharmacodynamic (PK/PD) models allowing for precision and personalized medicine. However, reliable and general in vitro-to-in vivo extrapolation and interspecies scaling methods are still lacking. Here, we developed a translational strategy for the anticancer drug oxaliplatin. Using ex vivo PK data in the whole blood of the mouse, rat, and human, a model representing the amount of platinum (Pt) in the plasma and in the red blood cells was designed and could faithfully fit each dataset independently. A "purely physiologically-based (PB)" scaling approach solely based on preclinical data failed to reproduce human observations, which were then included in the calibration. Investigating approaches in which one parameter was set as species-specific, whereas the others were computed by PB scaling laws, we concluded that allowing the Pt binding rate to plasma proteins to be species-specific permitted to closely fit all data, and guaranteed parameter identifiability. Such a strategy presenting the drawback of including all clinical datasets, we further identified a minimal subset of human data ensuring accurate model calibration. Next, a "whole body" model of oxaliplatin human PK was inferred from the ex vivo study. Its three remaining parameters were estimated, using one third of the available patient data. Remarkably, the model achieved a good fit to the training dataset and successfully reproduced the unseen observations. Such validation endorsed the legitimacy of our scaling methodology calling for its testing with other drugs.

Sex and Circadian Timing Modulate Oxaliplatin Hematological and Hematopoietic Toxicities.

Oxaliplatin was nearly twice as hematotoxic, with optimal circadian timing differing by 6 h, in women as compared to men with colorectal cancers. Hence, we investigated sex- and timing-related determinants of oxaliplatin hematopoietic toxicities in mice. Body-weight loss (BWL), blood cell counts, bone marrow cellularity (BMC) and seven flow-cytometry-monitored hematopoietic progenitor populations were evaluated 72 h after oxaliplatin chronotherapy administration (5 mg/kg). In control animals, circadian rhythms of circulating white blood cells showed a peak at ZT5 in both sexes, whereas BMC was maximum at ZT20 in males and ZT13h40 in females. All BM progenitor counts presented robust rhythms with phases around ZT3h30 in females, whereas only three of them rhythmically cycled in males with a ≈ -6 h phase shift. In treated females, chronotoxicity rhythms occurred in BWL, WBC, BMC and all BM progenitors with the best timing at ZT15, ZT21, ZT15h15 and ZT14h45, respectively. In males, almost no endpoints showed circadian rhythms, BWL and WBC toxicity being minimal, albeit with a substantial drop in BM progenitors. Increasing dose (10 mg/kg) in males induced circadian rhythms in BWL and WBC but not in BM endpoints. Our results suggest complex and sex-specific clock-controlled regulation of the hematopoietic system and its response to oxaliplatin.

Model learning to identify systemic regulators of the peripheral circadian clock.

MOTIVATION: Personalized medicine aims at providing patient-tailored therapeutics based on multi-type data toward improved treatment outcomes. Chronotherapy that consists in adapting drug administration to the patient's circadian rhythms may be improved by such approach. Recent clinical studies demonstrated large variability in patients' circadian coordination and optimal drug timing. Consequently, new eHealth platforms allow the monitoring of circadian biomarkers in individual patients through wearable technologies (rest-activity, body temperature), blood or salivary samples (melatonin, cortisol) and daily questionnaires (food intake, symptoms). A current clinical challenge involves designing a methodology predicting from circadian biomarkers the patient peripheral circadian clocks and associated optimal drug timing. The mammalian circadian timing system being largely conserved between mouse and humans yet with phase opposition, the study was developed using available mouse datasets. RESULTS: We investigated at the molecular scale the influence of systemic regulators (e.g. temperature, hormones) on peripheral clocks, through a model learning approach involving systems biology models based on ordinary differential equations. Using as prior knowledge our existing circadian clock model, we derived an approximation for the action of systemic regulators on the expression of three core-clock genes: Bmal1, Per2 and Rev-Erbα. These time profiles were then fitted with a population of models, based on linear regression. Best models involved a modulation of either Bmal1 or Per2 transcription most likely by temperature or nutrient exposure cycles. This agreed with biological knowledge on temperature-dependent control of Per2 transcription. The strengths of systemic regulations were found to be significantly different according to mouse sex and genetic background. AVAILABILITY AND IMPLEMENTATION: https://gitlab.inria.fr/julmarti/model-learning-mb21eccb. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Chronic circadian disruption modulates breast cancer stemness and immune microenvironment to drive metastasis in mice.

Breast cancer is the most common type of cancer worldwide and one of the major causes of cancer death in women. Epidemiological studies have established a link between night-shift work and increased cancer risk, suggesting that circadian disruption may play a role in carcinogenesis. Here, we aim to shed light on the effect of chronic jetlag (JL) on mammary tumour development. To do this, we use a mouse model of spontaneous mammary tumourigenesis and subject it to chronic circadian disruption. We observe that circadian disruption significantly increases cancer-cell dissemination and lung metastasis. It also enhances the stemness and tumour-initiating potential of tumour cells and creates an immunosuppressive shift in the tumour microenvironment. Finally, our results suggest that the use of a CXCR2 inhibitor could correct the effect of JL on cancer-cell dissemination and metastasis. Altogether, our data provide a conceptual framework to better understand and manage the effects of chronic circadian disruption on breast cancer progression.

Dosing time dependent in vitro pharmacodynamics of Everolimus despite a defective circadian clock.

Everolimus (EV), a rapamycin analogue mTOR inhibitor, is used in the clinic to treat Estrogen positive (ER+) breast cancer in order to avoid the resistance to hormonotherapy. Here, we investigated whether EV efficacy varied according to administration timing by using the ER+ breast cancer cell line MCF-7 as model system. Our results showed that instead of apoptosis, EV induced a G0/G1 phase blockage of MCF-7 cells. Following serum shock, MCF-7 cells displayed a statistically significant 24h rhythm of mammalian target of Rapamycin (mTOR) activity, but perturbed circadian clock genes oscillations. Interestingly, the different delivery schedule of EV presented different efficacy in G0/G1 phase blockage in serum shocked MCF-7 cells. Moreover, serum shock induced also a circadian-like oscillation in expression or activity of several important G1 phase progression proteins, such as Cyclin D1 and phosphorylated Retinoblastoma protein (RB). Inhibition mTOR activity by EV reduced Cyclin D1 and Cyclin D3 protein level as well as RB phosphorylation level. Taken together, the results indicated that serum shock synchronization induced a circadian oscillation in mTOR activity in MCF-7 cells, which rhythmically regulated the synthesis or phosphorylation of key G1 progression proteins, such as Cyclin D1 and phosphorylated RB, ultimately resulting in different G0/G1 blockage efficiency according to different EV administration timing.

Identification of Circadian Determinants of Cancer Chronotherapy through In Vitro Chronopharmacology and Mathematical Modeling.

Cancer chronotherapy aims at enhancing tolerability and efficacy of anticancer drugs through their delivery according to circadian clocks. However, mouse and patient data show that lifestyle, sex, genetics, drugs, and cancer can modify both host circadian clocks and metabolism pathways dynamics, and thus the optimal timing of drug administration. The mathematical modeling of chronopharmacology could indeed help moderate optimal timing according to patient-specific determinants. Here, we combine in vitro and in silico methods, in order to characterize the critical molecular pathways that drive the chronopharmacology of irinotecan, a topoisomerase I inhibitor with complex metabolism and known activity against colorectal cancer. Large transcription rhythms moderated drug bioactivation, detoxification, transport, and target in synchronized colorectal cancer cell cultures. These molecular rhythms translated into statistically significant changes in pharmacokinetics and pharmacodynamics according to in vitro circadian drug timing. The top-up of the multiple coordinated chronopharmacology pathways resulted in a four-fold difference in irinotecan-induced apoptosis according to drug timing. Irinotecan cytotoxicity was directly linked to clock gene BMAL1 expression: The least apoptosis resulted from drug exposure near BMAL1 mRNA nadir (P < 0.001), whereas clock silencing through siBMAL1 exposure ablated all the chronopharmacology mechanisms. Mathematical modeling highlighted circadian bioactivation and detoxification as the most critical determinants of irinotecan chronopharmacology. In vitro-in silico systems chronopharmacology is a new powerful methodology for identifying the main mechanisms at work in order to optimize circadian drug delivery.

Functional genomics identify Birc5/survivin as a candidate gene involved in the chronotoxicity of cyclin-dependent kinase inhibitors.

The circadian timing system orchestrates most of mammalian physiology and behavior in synchrony with the external light/dark cycle. This regulation is achieved through endogenous clocks present in virtually all body cells, where they control key cellular processes, including metabolism, transport, and the cell cycle. Consistently, it has been observed in preclinical cancer models that both the efficacy and toxicity of most chemotherapeutic drugs depend on their time of administration. To further explore the molecular basis underlying the link between the circadian timing system and the cellular response to anticancer drugs, we investigated the circadian transcriptome and CDK inhibitor toxicity in colon mucosa cells. We first show here that among 181 circadian transcripts, approximately 30% of them drive the cell cycle in the healthy mouse colon mucosa, with a majority peaking during the early resting phase. The identification of 26 mitotic genes within this cluster further indicated that the transcriptional coordination of mitosis by the circadian clock participates in the gating of cell division in this tissue. Subsequent selective siRNA-mediated silencing of these 26 targets revealed that low expression levels of the mitotic and anti-apoptotic gene Birc5/survivin significantly and specifically increased the sensitivity of colon epithelial cells to CDK inhibitors. By identifying Birc5/survivin as a potential determinant for the circadian modulation of CDK inhibitor toxicity, these data provide a mechanistic basis for the preclinical development of future CDK inhibitor-based chronotherapeutic strategies.

A circadian clock transcription model for the personalization of cancer chronotherapy.

Circadian timing of anticancer medications has improved treatment tolerability and efficacy several fold, yet with intersubject variability. Using three C57BL/6-based mouse strains of both sexes, we identified three chronotoxicity classes with distinct circadian toxicity patterns of irinotecan, a topoisomerase I inhibitor active against colorectal cancer. Liver and colon circadian 24-hour expression patterns of clock genes Rev-erbα and Bmal1 best discriminated these chronotoxicity classes, among 27 transcriptional 24-hour time series, according to sparse linear discriminant analysis. An 8-hour phase advance was found both for Rev-erbα and Bmal1 mRNA expressions and for irinotecan chronotoxicity in clock-altered Per2(m/m) mice. The application of a maximum-a-posteriori Bayesian inference method identified a linear model based on Rev-erbα and Bmal1 circadian expressions that accurately predicted for optimal irinotecan timing. The assessment of the Rev-erbα and Bmal1 regulatory transcription loop in the molecular clock could critically improve the tolerability of chemotherapy through a mathematical model-based determination of host-specific optimal timing.

A combined experimental and mathematical approach for molecular-based optimization of irinotecan circadian delivery.

Circadian timing largely modifies efficacy and toxicity of many anticancer drugs. Recent findings suggest that optimal circadian delivery patterns depend on the patient genetic background. We present here a combined experimental and mathematical approach for the design of chronomodulated administration schedules tailored to the patient molecular profile. As a proof of concept we optimized exposure of Caco-2 colon cancer cells to irinotecan (CPT11), a cytotoxic drug approved for the treatment of colorectal cancer. CPT11 was bioactivated into SN38 and its efflux was mediated by ATP-Binding-Cassette (ABC) transporters in Caco-2 cells. After cell synchronization with a serum shock defining Circadian Time (CT) 0, circadian rhythms with a period of 26 h 50 (SD 63 min) were observed in the mRNA expression of clock genes REV-ERBα, PER2, BMAL1, the drug target topoisomerase 1 (TOP1), the activation enzyme carboxylesterase 2 (CES2), the deactivation enzyme UDP-glucuronosyltransferase 1, polypeptide A1 (UGT1A1), and efflux transporters ABCB1, ABCC1, ABCC2 and ABCG2. DNA-bound TOP1 protein amount in presence of CPT11, a marker of the drug PD, also displayed circadian variations. A mathematical model of CPT11 molecular pharmacokinetics-pharmacodynamics (PK-PD) was designed and fitted to experimental data. It predicted that CPT11 bioactivation was the main determinant of CPT11 PD circadian rhythm. We then adopted the therapeutics strategy of maximizing efficacy in non-synchronized cells, considered as cancer cells, under a constraint of maximum toxicity in synchronized cells, representing healthy ones. We considered exposure schemes in the form of an initial concentration of CPT11 given at a particular CT, over a duration ranging from 1 to 27 h. For any dose of CPT11, optimal exposure durations varied from 3h40 to 7h10. Optimal schemes started between CT2h10 and CT2h30, a time interval corresponding to 1h30 to 1h50 before the nadir of CPT11 bioactivation rhythm in healthy cells.

Sex and dosing-time dependencies in irinotecan-induced circadian disruption.

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50 mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2 h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers.

Cancer inhibition through circadian reprogramming of tumor transcriptome with meal timing.

Circadian disruption accelerates cancer progression, whereas circadian reinforcement could halt it. Mice with P03 pancreatic adenocarcinoma (n = 77) were synchronized and fed ad libitum (AL) or with meal timing (MT) from Zeitgeber time (ZT) 2 to ZT6 with normal or fat diet. Tumor gene expression profiling was determined with DNA microarrays at endogenous circadian time (CT) 4 and CT16. Circadian mRNA expression patterns were determined for clock genes Rev-erbalpha, Per2, and Bmal1, cellular stress genes Hspa8 and Cirbp, and cyclin A2 gene Ccna2 in liver and tumor. The 24-hour patterns in telemetered rest-activity and body temperature and plasma corticosterone and insulin-like growth factor-I (IGF-I) were assessed. We showed that MT inhibited cancer growth by approximately 40% as compared with AL (P = 0.011) irrespective of calorie intake. Clock gene transcription remained arrhythmic in tumors irrespective of feeding schedule or diet. Yet, MT upregulated or downregulated the expression of 423 tumor genes, according to CT. Moreover, 36 genes involved in cellular stress, cell cycle, and metabolism were upregulated at one CT and downregulated 12 h apart. MT induced >10-fold circadian expression of Hspa8, Cirbp, and Ccna2 in tumors. Corticosterone or IGF-I patterns played no role in tumor growth inhibition. In contrast, MT consistently doubled the circadian amplitude of body temperature. Peak and trough respectively corresponded to peak expressions of Hspa8 and Cirbp in tumors. The reinforcement of the host circadian timing system with MT induced 24-hour rhythmic expression of critical genes in clock-deficient tumors, which translated into cancer growth inhibition. Targeting circadian clocks represents a novel potential challenge for cancer therapeutics.

Circadian timing in cancer treatments.

The circadian timing system is composed of molecular clocks, which drive 24-h changes in xenobiotic metabolism and detoxification, cell cycle events, DNA repair, apoptosis, and angiogenesis. The cellular circadian clocks are coordinated by endogenous physiological rhythms, so that they tick in synchrony in the host tissues that can be damaged by anticancer agents. As a result, circadian timing can modify 2- to 10-fold the tolerability of anticancer medications in experimental models and in cancer patients. Improved efficacy is also seen when drugs are given near their respective times of best tolerability, due to (a) inherently poor circadian entrainment of tumors and (b) persistent circadian entrainment of healthy tissues. Conversely, host clocks are disrupted whenever anticancer drugs are administered at their most toxic time. On the other hand, circadian disruption accelerates experimental and clinical cancer processes. Gender, circadian physiology, clock genes, and cell cycle critically affect outcome on cancer chronotherapeutics. Mathematical and systems biology approaches currently develop and integrate theoretical, experimental, and technological tools in order to further optimize and personalize the circadian administration of cancer treatments.

Characterization of SLC26A9, facilitation of Cl(-) transport by bicarbonate.

SLC26 family members are anionic transporters involved in Cl(-) and HCO(3)(-) absorption or secretion in epithelia. SLC26A9, preferentially expressed in the lung, is a poorly characterized member of this family. In this study, we investigated the transport properties of human SLC26A9 to determine its functional and pharmacological characteristics. SLC26A9 protein expression results in the appearance of an anionic current exhibiting an apparently linear current/voltage relationship and increases in (36)Cl influxes and effluxes. The sequences of conductivity, Cl(-) >I(-) > NO(3)(-) >/= gluconate > SO(4) (2-) and selectivity (P(x)/P(CI)), I(-) > NO(3)(-) > Cl(-) > gluconate > SO(4)(2-) are found. Cl(-) channel inhibitors DIDS and NS 3623 inhibit SLC26A9 associated currents while the specific CFTR inhibitor (CFTR(inh)-172) or glybenclamide has little effect. Elevation of intracellular cAMP (a CFTR activator) is also ineffective whereas increasing intracellular calcium blocks the SLC26A9 associated currents. The HCO(3)(-) conductance mediated by the SLC26A9 protein expression is low and no intracellular pHi changes are detectable under conditions favoring a Cl(-)/HCO(3)(-) exchange. However, the presence of HCO(3)(-)/CO(2) stimulates the Cl(-)-transporting activity of SLC26A9 in Xenopus laevis oocytes or SLC26A9-transduced COS-7 cells. As an important initial step in characterizing SLC26A9 function, we conclude that SLC26A9 is a Cl(-) channel and we suggest that HCO(3)(-) acts as a modulator of the channel. SLC26A9 physiological role in airway epithelia and its potential interaction with CFTR remain to be elucidated.

Altered Stra13 and Dec2 circadian gene expression in hypoxic cells.

The circadian system regulates rhythmically most of the mammalian physiology in synchrony with the environmental light/dark cycle. Alteration of circadian clock gene expression has been associated with tumour progression but the molecular links between the two mechanisms remain poorly defined. Here we show that Stra13 and Dec2, two circadian transcriptional regulators which play a crucial role in cell proliferation and apoptosis are overexpressed and no longer rhythmic in serum shocked fibroblasts treated with CoCl(2,) a substitute of hypoxia. This effect is associated with a loss of circadian expression of the clock genes Rev-erbalpha and Bmal1, and the clock-controlled gene Dbp. Consistently, cotransfection assays demonstrate that STRA13 and DEC2 both antagonize CLOCK:BMAL1 dependent transactivation of the Rev-erbalpha and Dbp promoters. Using a transplantable osteosarcoma tumour model, we show that hypoxia is associated with altered circadian expression of Stra13, Dec2, Rev-erbalpha, Bmal1 and Dbp in vivo. These observations collectively support the notion that overexpression of Stra13 and Dec2 links hypoxia signalling to altered circadian clock gene expression.

Involvement of the calcium-dependent proteolytic system in skeletal muscle aging.

Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern with high socio-economics implications. Although sarcopenia is well documented, the aetiology of this condition still remains poorly understood. Calpains are ubiquitous proteases regulated in part by a specific inhibitor, calpastatin. They are well known to have major implications in muscle growth and differentiation. The aim of the present study was to determine if this proteolytic system could be involved in the phenotype associated with sarcopenia. Calpains and calpastatin levels, subcellular distributions and activities were compared between muscles from 3 and 24 months old rats. Altogether, the results we obtained showed an overall increase in calpain activities associated with muscle aging. These findings suggest that the calcium-dependent proteolytic system is indeed involved in sarcopenia.

Enhancement of P2Y6-induced Cl- secretion by IL-13 and modulation of SK4 channels activity in human bronchial cells.

Expression of functional P2Y(6) receptors was demonstrated in primary cultures of human bronchial cells (NHBE cells). P2Y(6) receptors were located only on the apical membranes of NHBE cells. Their stimulation by UDP induced a chloride secretion (short-circuit current) reflected by the development of two I(sc) components (I(fast) and I(late)). A pharmacological characterization of those two I(sc) components showed the involvement of CaCC and CFTR channel activity in I(fast) and I(late) respectively. I(fast) was also found to be under control of basolateral SK4 channels. Indeed, inhibition of SK4 channels opening by clotrimazole dramatically reduced I(fast) amplitude. The epithelial ion transporting phenotype depends on the cellular state of differentiation. As previously reported, we observed that Ultroser G increased the epithelial tightness and Na(+)-transport capacity while IL-13 switch the epithelial ion transport phenotype from a Na(+)-absorbing to a Cl(-)-secreting one. In our study, we report for the first time a change in the K(+) cell permeability associated to IL-13-induced cell differentiation. IL-13 treatment increased the-resting K(+) permeability as well as the Ca(2+)-dependent K(+) permeability stimulated by UDP or ionomycin. SK4 channels activity, underlying the Ca(2+)-dependent K(+) permeability was in particular increased by IL-13. The on/off effect of IL-13 on P2Y(6)-induced Cl-secretion may help to identify the molecular determinants responsible for the CaCC channel activity.

Biologically active milli-calpain associated with caveolae is involved in a spatially compartmentalised signalling involving protein kinase C alpha and myristoylated alanine-rich C-kinase substrate (MARCKS).

We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.

Myristoylated alanine-rich C kinase substrate (MARCKS) is involved in myoblast fusion through its regulation by protein kinase Calpha and calpain proteolytic cleavage.

MARCKS (myristoylated alanine-rich C kinase substrate) is a major cytoskeletal protein substrate of PKC (protein kinase C) whose cellular functions are still unclear. However numerous studies have implicated MARCKS in the stabilization of cytoskeletal structures during cell differentiation. The present study was performed to investigate the potential role of Ca(2+)-dependent proteinases (calpains) during myogenesis via proteolysis of MARCKS. It was first demonstrated that MARCKS is a calpain substrate in vitro. Then, the subcellular expression of MARCKS was examined during the myogenesis process. Under such conditions, there was a significant decrease in MARCKS expression associated with the appearance of a 55 kDa proteolytic fragment at the time of intense fusion. The addition of calpastatin peptide, a specific calpain inhibitor, induced a significant decrease in the appearance of this fragment. Interestingly, MARCKS proteolysis was dependent of its phosphorylation by the conventional PKCalpha. Finally, ectopic expression of MARCKS significantly decreased the myoblast fusion process, while reduced expression of the protein with antisense oligonucleotides increased the fusion. Altogether, these data demonstrate that MARCKS proteolysis is necessary for the fusion of myoblasts and that cleavage of the protein by calpains is involved in this regulation.