Mast cell lines HMC‐1 and LAD2 in comparison with mature human skin mast cells – drastically reduced levels of tryptase and chymase in mast cell lines

Please cite this paper as: Mast cell lines HMC‐1 and LAD2 in comparison with mature human skin mast cells – drastically reduced levels of tryptase and chymase in mast cell lines. Experimental Dermatology 2010; 19 : 845–847. Abstract: To circumvent the costly isolation procedure associated with tissue mast cells (MC), two human MC lines, i.e. HMC‐1 and LAD2, are frequently employed, but their relation to mature MC is unknown. Here, we quantitatively assessed their expression of MC markers in direct comparison to skin MC (sMC). sMC expressed all lineage markers at highest and HMC‐1 cells at lowest levels. LAD2 cells expressed comparable high‐affinity IgE receptor α (FcεRIα) and FcεRIγ but less FcεRIβ than sMC and displayed slightly reduced, but robust FcεRI‐mediated histamine release. Only minor differences were found for total histamine content and c‐Kit expression. Huge, and to this level unexpected, differences were found for MC tryptase and chymase, with sMC >>> LAD2 > HMC‐1. Taken together, HMC‐1 cells represent very immature malignantly transformed MC, whereas LAD2 cells can be considered intermediately differentiated. Because of the minute levels of MC proteases, MC lines can serve as surrogates of tissue MC to a limited degree only.     

Association of paediatric mastocytosis with a polymorphism resulting in an amino acid substitution (M541L) in the transmembrane domain of c-KIT

Background The receptor tyrosine kinase c‐KIT plays a key role in normal mast cell development. Point mutations in c‐KIT have been associated with sporadic or familial mastocytosis. Objectives Two unrelated pairs of apparently identical twins affected by cutaneous mastocytosis attending the Mastocytosis Clinic at the Royal Children’s Hospital, Melbourne, provided an opportunity to assess the possible contribution of c‐KIT germline mutations or polymorphisms in this disease. Methods Tissue biopsy, blood and/or buccal swab specimens were collected from 10 children with mastocytosis. To detect germline mutations/polymorphisms in c‐KIT, we studied all coding exons by denaturing high pressure liquid chromatography. Exons showing mismatches were examined by direct sequencing. The influence of the substitution identified was further examined by expressing the variant form of c‐KIT in factor‐dependent FDC‐P1 cells. Results In both pairs of twins, a heterozygous ATG to CTG transition in codon 541 was observed, resulting in the substitution of a methionine residue in the transmembrane domain by leucine (M541L). In each case, one parent was also heterozygous for this allele. Expression of M541L KIT in FDC‐P1 cells enabled them to grow in human KIT ligand (stem cell factor, SCF) but did not confer factor independence. Compared with cells expressing wild‐type KIT at a similar level, M541L KIT‐expressing cells displayed enhanced growth at low levels of SCF, and heightened sensitivity to the KIT inhibitor, imatinib mesylate. Conclusions The data suggest that the single nucleotide polymorphism resulting in the substitution M541L may predispose to paediatric mastocytosis.     

Increased mast cell expression of PAR‐2 in skin inflammatory diseases and release of IL‐8 upon PAR‐2 activation

Please cite this paper as: Increased mast cell expression of PAR‐2 in skin inflammatory diseases and release of IL‐8 upon PAR‐2 activation. Experimental Dermatology 2010; 19: 117–122. Abstract: Mast cells are increasingly present in the lesional skin of chronic skin inflammatory diseases including psoriasis and basal cell carcinoma (BCC). It has previously been shown that proteinase‐activated receptor (PAR)‐2 is expressed by mast cells, and tryptase is a potent activator of this receptor. In this study, skin biopsies from both healthy‐looking and lesional skin of patients with psoriasis and superficial spreading BCC were collected and the expression of PAR‐2 immunoreactivity in tryptase‐positive mast cells was analysed. PAR‐2 expression was confirmed in vitro in different mast cell populations. Cord‐blood derived mast cells (CBMC) were stimulated with a PAR‐2 activating peptide, 2‐furoyl‐LIGRLO‐NH₂. Consequently, IL‐8 and histamine production was analysed in the supernatants. We observed a significant increase in the percentage of mast cells expressing PAR‐2 in the lesional skin of psoriasis and BCC patients compared with the healthy‐looking skin. HMC‐1.2, LAD‐2 and CBMC mast cells all expressed PAR‐2 both intracellularly and on the cell surface. CBMC activation with the PAR‐2 activating peptide resulted in an increased secretion of IL‐8, but no histamine release was observed. Furthermore, both PAR‐2 and IL‐8 were co‐localized to the same tryptase‐positive mast cells in the lesional BCC skin. These results show that mast cells express increased levels of PAR‐2 in chronic skin inflammation. Also, mast cells can be activated by a PAR‐2 agonist to secrete IL‐8, a chemokine which can contribute to the progress of inflammation.     

NKp46 regulates the production of serine proteases and IL‐22 in human mast cells in urticaria pigmentosa

NKp46 (natural cytotoxic receptor 1/CD335) is expressed on natural killer cells and Th2‐type innate lymphocytes. However, NKp46 expression in human mast cells has not yet been reported. Here, we explored the expression of, and possible role played by, NKp46 in such cells. NKp46 protein was expressed in human mast cells in urticaria pigmentosa principally of the tryptase‐positive/chymase‐negative type (MCT), but not in human non‐neoplastic skin mast cells of the tryptase‐positive/chymase‐positive (MCTC) type. NKp46 expression was also evident in the human neoplastic mast cell line HMC1.2. NKp46 knockdown changed the phenotype of this cell line from MCT to MCTC and downregulated GrB production, but did not influence IL‐22 production. An agonistic anti‐NKp46 antibody upregulated production of GrB and IL‐22, but did not change the MCT‐like phenotype of HMC1.2 cells. NKp46 was thus involved in the production of serine proteases and IL‐22 in human mast cells.     

Ultraviolet irradiation induces apoptosis in human immature, but not in skin mast cells

As diverse pruritic cutaneous diseases respond to ultraviolet treatment, we have examined whether ultraviolet light is capable of inducing apoptosis in mast cells. Human mast cell line 1 (HMC1) derived from a patient with malignant mastocytosis and purified skin mast cells were irradiated with single doses of ultraviolet B or ultraviolet A1, or pretreated with 8-methoxypsoralen prior to ultraviolet A1 exposure. After 0 to 48 h of incubation, the percentage of apoptotic and dead cells was assessed. In HMC1 cells, morphologic features of apoptosis were further evaluated by electron microscopy. All ultraviolet treatment induced apoptosis of HMC1 cells in a time- and dose-dependent manner. Apoptosis was associated with activation of caspase-3, release of cytochrome C, cleavage of poly(ADP-ribose)-polymerase, and nuclear accumulation of p53. In contrast, resting skin mast cells were resistant to ultraviolet light induced apoptosis. After incubation with stem cell factor and interleukin-4 for 2 wk, however, slowly proliferating skin mast cells also underwent apoptosis in response to ultraviolet light. In conclusion, these data demonstrate that ultraviolet light directly affects mast cells, but mainly aims at the proliferating mast cells as found in mastocytosis and mast cell dependent pruritic diseases, where increased numbers are observed due to the recruitment mast cell precursors from the blood.     

Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE‐independent mast cell degranulation elicited by neuromuscular blocking agents

Please cite this paper as: Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE‐independent mast cell degranulation elicited by neuromuscular blocking agents. Experimental Dermatology 2010; 19: 302–304. Abstract: Women predominate in the anaphylactic reactions to neuromuscular blocking agents (NMBA). The expression of oestrogen receptors has been demonstrated in mast cells and oestrogen treatment can enhance mast cell degranulation, but the influence of androgens remains largely unclear. Our immunocytochemical study showed the expression of androgen receptor (AR) in mast cells isolated from human foreskin as well as in two human mast cell lines, HMC‐1 and LAD2. The amount of AR was most abundant in human skin mast cells as determined by real‐time polymerase chain reaction analysis. Treatment of the HMC‐1 mast cells with testosterone or 17β‐oestradiol, alone or in combination with different NMBA, did not affect mast cell degranulation as measured by the release of β‐hexosaminidase. Our study shows for the first time the expression of AR in human skin mast cells. Further studies using primary human mast cell cultures are needed to understand whether and how sex hormones can influence mast cell activation.     

BRAF, HRAS, KRAS, NRAS and CDKN2A genes analysis in cultured melanocytes used for vitiligo treatment

Background Cultured pigment cells are increasingly being used in the treatment of stable vitiligo. The melanocyte growth media contain synthetic and human recombinant mitogenic factors. High concentration of growth factors, increased melanin biosynthesis, and the rapid cell cycle progression may lead to the genetic material damage and the initiation of melanocyte malignant transformation in cell culture conditions. Mutations of genes of the RAS/RAF/MEK/ERK signaling pathway and CDKN2A gene are often found in the early stages of melanoma development. 12‐O‐Tetradecanoyl‐phorbol‐13‐acetate (TPA)/phorbol 12‐myristate 13‐acetate (PMA) is considered to be an oncogenic factor, but there is no evidence that it is responsible for melanomagenesis initiation. The goal of this research was to assess the risk of the development of mutations in selected genes of the RAS/RAF/MEK/ERK signaling pathway and CDKN2A gene during the culturing of pigment cells in various growth media. Methods Three‐hundred melanocyte cultures were established in 10 various growth media. The population doubling time of cultured cells was calculated for all the tested growth media. Cytogenetic analysis was carried out on the HRAS (exon 1 and 2), KRAS (exon 1 and 2), NRAS (exon 1 and 2), BRAF (exon 11 and 15), and CDKN2A (exon 1) genes. Results Our study revealed that TPA and high concentrations of other growth factors intensify the proliferation of pigment cells, without the risk of damage to the analyzed genes. Conclusions It is necessary to carry out further similar studies on other signaling pathways to confirm cultured melanocytes transplantation safety.     

Dimethylfumarate induces apoptosis in human mast cells

Mast cells modulate autoimmune diseases such as psoriasis and multiple sclerosis. Fumaric acid esters (FAEs) are widely used for the treatment of psoriasis, and dimethylfumarate (DMF) has recently been approved for multiple sclerosis. In this study, we analysed the cytotoxic effect of FAEs on human mast cells. Specifically, cell death was analysed in the human mast cell line HMC‐1 and in primary cord blood‐derived mast cells (CBMCs) after incubation with fumaric acid (FA), monomethylfumarate (MMF), DMF and calcium bis(monomethylfumarate) (Ca‐MF). Our data show that only DMF potently induces apoptotic cell death in HMC‐1 cells and CBMCs. DMF‐mediated apoptosis was associated with increased expression of Bax and Bak and activation of caspase‐9 and caspase‐6. Interestingly, DMF also enhanced the sensitivity of CBMCs towards TRAIL‐ and dexamethasone‐induced apoptosis. These findings demonstrate for the first time that DMF induces apoptosis of human mast cells, primarily via the mitochondrial apoptotic pathway. Our study contributes to the understanding of the beneficial effects of FAEs in autoimmune diseases and provides a rationale for exploiting FAEs for other diseases associated with mast cells.     

A new germline mutation in KIT associated with diffuse cutaneous mastocytosis in a Chinese family

Diffuse cutaneous mastocytosis (DCM) is an extremely rare disease characterized by massive proliferation of mast cells infiltrating the entire skin. We report a Chinese family with indolent DCM, and detection of a new germline KIT mutation located in the fifth immunoglobulin‐like loop of the KIT protein, which probably results in a gain‐of‐function effect and consequent overactivation of mast cells. Our report expands the knowledge of correlations between the genotype of KIT mutations and the phenotype of DCM.     

Is Mcl‐1L the new anti‐apoptotic effector of B‐RAFV600E in melanoma?

A recent report has unveiled a novel mechanism by which oncogenic BRAF signalling might trigger apoptotic resistance in melanoma by selectively affecting the expression of Bcl‐2 family member Mcl‐1L (Exp Dermatol 2013: 22 : 767). Correlation of Mcl‐1 splice variants and B‐RAF mutational status was determined in a panel of melanoma cell lines. In vivo validation of this mechanism, which is supported by recent literature, might provide novel therapeutic approaches such as the use of targeted Mcl‐1 inhibitors to improve outcome in melanoma.     

Ablation of human skin mast cells in situ by lysosomotropic agents

Mast cells are known to have a detrimental impact on numerous types of inflammatory skin diseases such as contact dermatitis, atopic eczema and cutaneous mastocytosis. Regimens that dampen skin mast cell‐mediated activities can thus offer an attractive therapeutic option under such circumstances. As mast cells are known to secrete a large array of potentially pathogenic compounds, both from preformed stores in secretory lysosomes (granules) and after de novo synthesis, mere inhibition of degranulation or interference with individual mast cell mediators may not be sufficient to provide an effective blockade of harmful mast cell activities. An alternative strategy may therefore be to locally reduce skin mast cell numbers. Here, we explored the possibility of using lysosomotropic agents for this purpose, appreciating the fact that mast cell granules contain bioactive compounds prone to trigger apoptosis if released into the cytosolic compartment. Based on this principle, we show that incubation of human skin punch biopsies with the lysosomotropic agents siramesine or Leu‐Leu methyl ester preferably ablated the mast cell population, without causing any gross adverse effects on the skin morphology. Subsequent analysis revealed that mast cells treated with lysosomotropic agents predominantly underwent apoptotic rather than necrotic cell death. In summary, this study raises the possibility of using lysosomotropic agents as a novel approach to targeting deleterious mast cell populations in cutaneous mastocytosis and other skin disorders negatively influenced by mast cells.     

Sustained improvement in urticaria pigmentosa and pruritus in a case of indolent systemic mastocytosis treated with cladribine

Systemic mastocytosis (SM) is a myeloproliferative disorder, characterized by a clonal proliferation of abnormal mast cells accumulating in internal organs and sometimes in the skin, leading to cutaneous and systemic symptoms. Mutations within the gene KIT, which encodes the receptor tyrosine kinase (KIT) on mast cells, is found in most patients with SM. We report a case of a 62‐year‐old woman presenting with a pruritic rash on her limbs and trunk. Several years later she developed gastrointestinal symptoms, associated with raised serum tryptase. Skin and bone marrow biopsies confirmed a diagnosis of SM, initially presenting with urticaria pigmentosa. Responses to multiple therapies, including potent topical steroids, oral antihistamines, phototherapy and the tyrosine kinase inhibitor, nilotinib, were inadequate. Treatment with cladribine (2‐chlorodeoxyadenosine) produced a marked and sustained reduction in her symptoms and serum tryptase level.     

Evidence for altered mast cell proliferation and apoptosis in cutaneous mastocytosis

BACKGROUND: Mastocytosis presents as a focal or generalized increase of mast cells, particularly in the skin, but also in other organs. Activating mutations of KIT (formerly c-kit), the receptor of the mast cell growth factor stem cell factor (SCF), appear to play a key role in the pathogenesis of sporadic adult onset mastocytosis. However, these mutations are not present in childhood-onset and familial mastocytosis and also fail to explain the heterogeneity of adult-onset disease. Other factors such as prolonged survival of mast cells may therefore participate in causing and modulating the pathological increase of mast cells in mastocytosis. OBJECTIVES: To examine the expression of proliferation and apoptosis markers in the mast cells of cutaneous mastocytosis lesions in order to gain further insight into the pathogenesis of mastocytosis. METHODS: Lesional cutaneous biopsies from eight infants with solitary mastocytomas, five children with multiple mastocytomas, 11 children with generalized urticaria pigmentosa, 12 adults with urticaria pigmentosa, and skin from seven normal controls were used in this study. Serial sections were stained with toluidine blue to quantify mast cell numbers and with antibodies against the proliferation marker Ki67 protein, the tumour suppressor protein p53, and the inhibitor of cyclins and cyclin-dependent kinases p21WAF1/CIP1, using the alkaline phosphatase antialkaline phosphatase technique. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) method was used to assess apoptosis. RESULTS: Cutaneous mast cell counts were significantly increased in all patient sections, particularly in childhood lesions, and similarly, a small but significant increase of proliferation was found in the lesional mast cells of all patients. Enhanced mast cell numbers and proliferation was associated with a significant decrease of TUNEL staining, particularly in mastocytomas. p53 expression was highly variable, with an overall significant increase in all patient skin mast cells, whereas p21 expression was barely observed at all. CONCLUSIONS: These findings further support the concept that an imbalance of mast cell proliferation and apoptosis is prevalent in mastocytosis lesions that may account in part for the increased focal mast cell accumulation in this condition.     

Combined targeting of MAPK and AKT signalling pathways is a promising strategy for melanoma treatment

BACKGROUND: In melanoma, several signalling pathways are constitutively activated. Among them, the RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT (AKT) signalling pathways are activated through multiple mechanisms and appear to play a major role in melanoma development and progression. OBJECTIVES: In this study, we examined whether targeting the MAPK and/or AKT signalling pathways would have therapeutic effects against melanoma. METHODS: Using a panel of pharmacological inhibitors (BAY 43-9006, PD98059, U0126, wortmannin, LY294002) we inhibited the MAPK and AKT signalling pathways at different levels and evaluated the effects on growth, survival and invasion of melanoma cells in monolayer and organotypic skin culture. RESULTS: Antiproliferative and proapoptotic effects of inhibitors alone in monolayer culture were disappointing and varied among the different cell lines. In contrast, combined targeting of the MAPK and AKT signalling pathways significantly inhibited growth and enhanced apoptosis in monolayer culture. To verify our data in a more physiological context we incorporated melanoma cells into regenerated human skin mimicking the microenvironment of human melanoma. Combinations of MAPK and AKT inhibitors completely suppressed invasive tumour growth of melanoma cells in regenerated human skin. CONCLUSIONS: Combined targeting of MAPK and AKT signalling pathways is a promising strategy for melanoma treatment and should encourage further in-depth investigations.     

The efficacy of omalizumab in Cutaneous Mastocytosis: A case series

Background: Mastocytosis describes a heterogeneous group of disorders arising from a clonal proliferation of mast cells. Given the lack of curative treatments for the cutaneous form, there is a significant need for superior therapies. Omalizumab is a recombinant DNA‐derived humanized IgG monoclonal antibody that selectively binds to human immunoglobulin E (IgE). It represents a potential treatment for the management of cutaneous mastocytosis, which currently has no standard treatment. Methods: Two patients were treated with subcutaneous omalizumab 300 mg every 4 weeks. Discussion: Patient 1 experienced 50% reduction in cutaneous infiltration and moderate improvement in pruritus. Patient 2 underwent 90% complete clearance of cutaneous lesions and reported full resolution of pruritus. The median duration of treatment was 24 weeks and time to response was 8 weeks. No significant changes in tryptase levels were observed. Both patients experienced injection site reactions. Conclusion: We provide evidence from two cases supporting the efficacy of IgE‐mediated therapy in the treatment of cutaneous mastocytosis. Even at a higher‐than‐standard dose (300 mg vs. 150 mg), the drug was well‐tolerated. As we await the results of pivotal clinical trials, omalizumab appears to be a promising treatment option in patients with cutaneous mastocytosis unresponsive to traditional therapies.     

Ultrastructural morphometric analysis of human mast cells in normal skin and pathological cutaneous lesions

Electron micrographs of human mast cells in normal neonatal and adult skin and in cutaneous lesions of basal cell carcinoma (BCC), hemangioma and mastocytosis were assessed by morphometric analysis. Using this quantitative histologic approach, adult skin mast cells were found to be significantly larger (47.7 microns 2 +/- 2.4 SEM vs. 38.3 microns 2 +/- 1.8 SEM, p less than or equal to 0.001) and have larger granules (0.63 micron +/- .02 SEM vs. 0.53 micron +/- .02 SEM, p less than or equal to 0.001) than infant mast cells while both mast cell populations had comparable nuclear sizes (13.7 microns 2 +/- 0.9 SEM vs. 14.3 microns 2 +/- 0.8 SEM) and numbers of cytoplasmic granules (72 +/- 4.0 SEM vs. 66 +/- 4.0 SEM). Morphometric analysis of mast cell infiltrates in the adult skin lesions of BCC and hemangioma revealed that these cells were larger than neonatal mast cells but were similar to normal adult controls. Cutaneous mast cells from 2 mastocytosis patients, however, had significantly larger mean cell surface areas (78.0 microns 2 +/- 3.4 SEM and 70.6 microns 2 +/- 3.2 SEM, p less than or equal to 0.001), nuclear areas (20.8 microns 2 +/- 1.1 SEM and 21.3 microns 2 +/- 1.2 SEM p less than or equal to 0.001) and granule diameters (0.82 micron +/- 0.4 SEM and 0.83 micron +/- .03 SEM, p less than or equal to 0.001) when compared with mast cells in normal adult skin and in the other pathologic lesions. No difference in the total number of cytoplasmic granules was observed in the different mast cell populations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mast cell chymase in experimentally induced psoriasis

Mast cell chymase can have a pro‐inflammatory or an immunosuppressive function in psoriasis, but the outcome may depend on the level of chymase activity. Therefore, mast cells showing chymase activity (Chy_act) and immunoreactivity (Chy_prot) were studied during the Köbner reaction (0 days, 2 h, 1 day, 3 days and 7 days) of psoriasis induced by the tape‐stripping technique. Also, the effect of recombinant human chymase (rh‐chymase) or human LAD2 mast cells (LAD2) on the ³H‐thymidine uptake of psoriatic peripheral blood mononuclear cells (PBMC) or total T cells was studied. The Chy_act/Chy_prot ratio tended to be higher in all time‐point biopsies in the Köbner‐negative (n = 10) than ‐positive (n = 8) group (P = 0.073), although chymase activity decreased significantly at 2 h to 1 day only in the Köbner‐negative group. rh‐chymase (0.05–0.5 μg/mL) stimulated to a varying extent PBMC in eight out of nine cultures, but in all cultures 5 μg/mL rh‐chymase turned the stimulation towards inhibition. The effect of rh‐chymase on T cells varied from stimulation to inhibition, but in 11 of 15 cultures rh‐chymase, at least at 5 μg/mL, produced a change to inhibition. In co‐cultures, LAD2 inhibited PBMC in the absence of soybean trypsin inhibitor (SBTI). In the presence of SBTI, LAD2 stimulated PBMC in the majority of seven cultures. In summary, the psoriatic immunopathogenesis may be promoted at low, but controlled at high, activity status of chymase.     

Basic fibroblast growth factor stimulates the proliferation of human dermal fibroblasts via the ERK1/2 and JNK pathways

Background Basic fibroblast growth factor (bFGF, FGF‐2) has been described as a multipotent cytokine that regulates cell growth as well as differentiation, matrix composition, chemotaxis, cell adhesion and migration in numerous cell types. It is known that bFGF stimulates proliferation of cultured fibroblasts. However, the detailed mechanism of fibroblast proliferation induced by bFGF in vitro still remains to be elucidated. Objectives We investigated the precise effects of bFGF on fibroblast proliferation and the signalling pathways responsible for bFGF‐induced proliferation in cultured human dermal fibroblasts (HDFs). Methods HDFs were cultured with bFGF in the presence or absence of specific inhibitors against MAPK signalling pathways including ERK, JNK and p38. The number of cells was counted and immunoblotting findings were examined for the activation of ERK1/2 and JNK. Furthermore, the inhibitory effects of ERK1, ERK2 and JNK1 were proven by the transfection of siRNA. Results bFGF increased the number of HDFs in a dose‐ and time‐dependent manner. The bFGF‐induced proliferation was suppressed by the MEK inhibitors PD98059 and U0126, and the JNK inhibitor SP600125. bFGF increased the phosphorylation levels of ERK1/2 and JNK1. Treatment with ERK1, ERK2 or JNK1 siRNA significantly inhibited bFGF‐induced proliferation. Conclusions This study indicates that ERK1/2 and JNK pathways play an important role in the bFGF‐mediated effect in HDFs. This study also suggests that controlling ERK1/2 and/or JNK signalling may therefore be a new therapeutic approach for the treatment of chronic and untreatable skin ulcers.     

BCL2 and BCLxL are key determinants of resistance to antitubulin chemotherapeutics in melanoma cells

Malignant melanoma is refractory to various chemotherapeutics including antitubulin agents such as paclitaxel. Previous studies have suggested a link between βIII‐tubulin overexpression and paclitaxel resistance through alterations in the properties of the mitotic spindle. We found that paclitaxel treatment induced temporary mitotic arrest in 7 melanoma cell lines irrespective of the βIII‐tubulin level, suggesting that βIII‐tubulin had no significant influence on spindle properties. On the other hand, the amount of BCL2, an anti‐apoptotic protein, was well correlated with paclitaxel resistance. Treatment of the paclitaxel‐resistant cell lines with ABT‐737, an inhibitor of BCL2 and BCLxL, or simultaneous knock‐down of BCL2 and BCLxL dramatically increased the cells’ sensitivity, while knock‐down of MCL1, another member of the BCL2 family, had only a minimal effect. Our results suggest that the paclitaxel sensitivity of melanoma cells is attributable to apoptosis susceptibility rather than a change in spindle properties and that BCL2 and BCLxL play a pivotal role in the former.