Letter to the Editor
7 Poeggeler B, Schulz C, Pappolla M A et al. Exp Dermatol 2010: 19: 12–18. 8 Sumikawa Y, Inui S, Nakajima T et al. Exp Dermatol 2014: 23: 27–32. 9 Ibrahim L, Wright E A. J Embryol Exp Morphol 1975: 33: 831–844. 10 Van Harmelen V, Reynisdottir S, Eriksson P et al. Diabetes 1998: 47: 913–917.
Supporting Information Additional supporting data may be found in the supplementary information of this article. Figure S1. A cross section of mouse dorsal skin is showing the dermal white adipose tissue (DWAT) below dermis. Figure S2. The levels of leptin, adiponectin, BMP-2, and BMP-4 mRNA expressed by adipocytes and preadipocytes were measured with QPCR. Preadipocytes were isolated from DWAT of 5-week-old female C56BL/6 mice.
Figure S3. The longitudinal sections of anagen vibrissa hair follicles demonstrate where DWAT surrounds the hair follicle. Figure S4. (a) A schematic representation of expression patterns of adipose-derived factors throughout hair cycle stages from hair follicles and dermal adipose tissue. Dashed lines are hypothetical (yet to be determined). ‘C’: catagen; ‘EA’: early anagen; ‘ET’: early telogen; ‘LA’: late anagen; ‘LT’: late telogen. (b) A diagrammatic representation of the results collective from the literature showing the effects of paracrines, i.e., leptin, adiponectin, BMP-2, BMP-4, and PDGF, from skin adipocytes or preadipocytes on hair follicle growth. ‘?’: stimulatory; ‘┤’: inhibitory. Dashed lines are ‘hypothetical’. In telogen, leptin from adipocytes (reference 1) and PDGF from preadipocytes (reference 2) serve as anagen inducers by activating hair follicle epithelial stem cells and, therefore, hair growth. In contrast, BMP-2 and BMP-4 from adipocytes inhibits stem
cell activation and, therefore, blocking anagen induction (reference 3). The size of dermal adipose tissue is largely reduced in telogen, which suggests that a regulatory feedback loop from the hair follicle might exist in regulating adipose tissue metabolism. In anagen, leptin from adipocytes may contribute to the inhibitory effect which the adipocytes have on hair growth through possibly leptin receptors on the epithelial stem cells (this study). Although with mechanism unknown, the preadipocytes also exhibit an inhibitory effect on hair growth on anagen phase hair follicle (this study). On the other hand, adiponectin (derived from adipocytes) seems to promote hair growth at this stage (reference 4). The size of dermal adipose tissue increases during this time indicating that a positive signal might exist from the hair follicle to stimulate the expansion of the adipose tissue. Data S1. Material and methods. Data S2. Acknowledgements.
DOI: 10.1111/exd.12567
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
Characterization of the novel Sezary lymphoma cell line BKP1 John Boudjarane1,2, Arnaud Essaydi3, Laure Farnault2,4, Cornel Popovici1, Marina Lafage-Pochitaloff1,2, €l Berda-Haddad5, Romaric Lacroix5, Corinne Nicolino-Brunet5, The re se Le Treut6, Nathalie Beaufils3, Yae 1,2 2,3,7 2,8 2,4,8 s and Re gis Costello le ne Zattara , Jean Gabert , Brigitte Kahn-Perle He 1 Departement de Genetique Medicale, H^ opital La Timone, Assistance Publique-H^ opitaux de Marseille, Marseille, France; 2Aix-Marseille Universite, opital Nord, Marseille, France; 4Service d’Hematologie, H^ opital La Marseille, France; 3Laboratoire de Biochimie et de Biologie Moleculaire, H^ Conception, Marseille, France; 5Laboratoire d’Hematologie, H^ opital La Conception, Marseille, France; 6Laboratoire d’Hematologie, H^ opital Nord, Marseille, France; 7Unite INSERM 1072, Marseille, France; 8Technologies Avancees pour la Genomique et la Clinique (TAGC)/unite INSERM U1090, Marseille, France Correspondence: Regis T Costello, Service d’Hematologie, Centre Hospitalier Universitaire La Conception, 147 Boulevard Baille, 13005 Marseille, France, Tel.: +(33)-4-91-38-41-50, Fax: +(33)-4-91-38-41-54, e-mail: [email protected]
Abstract: Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of lymphomas primarily involving the skin. The most common types are mycosis fungoides (MF) and Sezary Syndrome (SS). We report a novel long-term fast-growing SS line termed BKP1 that was characterized by flow cytometry (FC), conventional and molecular cytogenetic [FISH/multi-FISH together with array comparative genomic hybridization (aCGH)]. FC immunophenotype of the BKP1 is CD2+CD5+CD3+ CD4+CD8 CD7 CD25 CD26 CD30 CD158k+. The TCRc characterization of BKP1 by PCR identified a clonal rearrangement. The conventional cytogenetic and Multi-FISH analysis showed complex chromosomal rearrangements. aCGH
analysis highlighted the loss of genes involved in cell cycle control, in immune response (HLA, complement complex) and DNA damage repair mechanisms. The BKP1 is another lymphoma cell line thoroughly characterized that can be a valuable tool for both basic and applied research such as identification of deregulated genes and/or pathways and screening for new antilymphoma drugs.
Introduction
only four SS cell lines [Se-Ax (3), HUT78, Pno (4) and Cou (5)] and one MF cell line [MyLA 2059 (6)] have been published.
Cutaneous T-cell lymphomas are a heterogeneous group primarily involving the skin (1). Most common types are MF and SS that are characterized by invasion of skin, lymph nodes or blood (SS) by CD4+ T cells phenotypically close to memory-effector T cells for MF and central-memory T cells for SS (2). Treatment of SS is disappointing even though a large number of drugs have been tested in clinical trials. The possibility to test in vitro and in vivo (animal models) SS cells is an important issue to identify novel drugs. So far,
60
Key words: cutaneous lymphoma – karyotype – T-cell lymphoma
Accepted for publication 10 October 2014
Questions addressed We report a novel long-term fast-growing SS line termed BKP1 that was characterized by FC phenotype, conventional karyotype completed by fluorescent in situ hybridization (FISH)/multi-FISH study together with aCGH study. This study represents the most comprehensive characterization, to our knowledge, of a novel SS cell line.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 55–80
Letter to the Editor
Experimental design Peripheral blood from SS patient was obtained after informed consent. Mononucleated cells were isolated by Ficoll-Paque density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) and cultured in 24-well culture plate in RPMI medium (BioWhittaker, Verviers, Belgium) containing 10% foetal calf serum (FCS, Perbio Science, Brebieres, France), interleukin (IL)-7 10 ng/ml (Genzyme, Cambridge, UK) and anti-CD28 monoclonal antibody 248 (D Olive, 1/400 dilution) at a 106 cells/ml initial concentration.
Results Flow cytometry immunophenotyping of both patient’s PB at diagnosis and BKP1 (three phenotypic assays after 1, 3 and 6 months culture) showed the same expression profile: CD2+CD5+CD3+CD4+CD8 CD7 CD25 CD26 CD30 CD158k+ (Fig. 1). FC Vb repertoire analyses were performed on patient’s PB at diagnosis and on the BKP1 (two phenotypic assays after 1 and 6 months culture). FC Vb repertoire indicates the clonal expansion of T cells (Fig. 1). Analysis of PCR TCR c VJ product showed the two clonal products (one in each tube A and B) in both patient’s PB at diagnosis, BKP1 (6 months culture; Figure S1) and skin biopsy with the same following sizes: tube A, an unique fragment of 257 base pairs, tube B, an unique fragment of 190 base pairs. Sequencing of the PCR products and in silico analysis using IMGT database allowed the identification of the TCRc VJ rearrangement (Table S1).
(a)
The conventional R-bands cytogenetic analysis of cell line showed a complex karyotype with numerical and structural abnormalities that was completed by multi-FISH analysis (Figure S2 and Table S2). Comprehensive genomic profile of gains and losses for BKP1 was generated with aCGH (Figure S3 and Table S3). The aCGH profile of BKP1 confirmed the complex patterns of rearrangements previously demonstrated with multi-FISH and conventional cytogenetics. We do not observe any anomaly in 6q23.3 and 11p15.5 regions. These two regions contain respectively AHI1 and CDKN1C genes, described to have a role in CTCL progression (7).
Discussion We show that BKP1 expresses compatible immunophenotype with Sezary cells. Indeed, we found CD158k expression which is recurrently expressed by neoplastic cells in SS (8) and CD30 and CD26 are negative. We identified with the aCGH genetic defects on genes involved in carcinogenesis of MF and SS. Firstly, we have detected the deletion of CDKN2A (p16) and CDKN2B (p15) genes, leading to a loss of cell cycle control. This anomaly is frequently observed in the late stages of CTCL and is associated with poor prognosis (9). Zhang et al. (10) showed a decreased expression of p16 in the skin lesions of MF and SS late stage evoking a probable proliferative advantage to cells having lost the expression of p16. We also identified in BKP1 cell line a deletion of FANCC, FANCM and XPA. These three genes are involved in the repair of DNA damage so that their deletions could lead to genomic instability. We observed in the BKP1 cell line a duplication 17q that is a recurrent (17–28%) abnormality considered as an early pathogenic event that is involved in SS cell proliferation advantage (11). The 17q region includes STAT3 and STAT5 genes, which are transcription factors involved in oncogenesis since constitutively activated in CTCL (12). These transcription factors activate the Ras/MEK/ERK and PI3K/Akt pathways, suggesting clues for the use of metabolic inhibitors for treatment of MF and SS. BKP1 also presents a gain of REL gene (2p13) and loss of IKBA (14q13). Both genes are involved in the NFKB pathway, REL as an activator and IKBA as an inhibitor. The gain of an activator and loss of an inhibitor of NFKB signalling pathway could contribute to the constitutive activation of this pathway. Constitutive activation of
(b) Table 1. Comparison of BKP1 cell line aCGH abnormalities with those of the literature
Figure 1. Flow cytometry analysis of BKP1 cell line. (a) Analysis of the TcR Vbeta repertoire by flow cytometry. The Sezary cells were analysed using a TcR Vbeta panel covering 70% of the TcR Vbeta repertoire. In each quadrant is indicated the Vbeta reactivity tested. (b) Flow cytometry analysis of KIRDL2/CD158k, DDIV/CD26 (R&D Systems Minneapolis Europe) and CD30 (Beckman Coulter). There are singleparameter histograms with: 1 On ● ● ●
the left: Isotypic PE control (purple) with a mean fluorescence intensity (MFI) of 0,13 CD26 (green) with MFI of 0,20 CD158k (blue) with MFI of 0,83
2 On the right: ● CD30 (red) with MFI 0,10.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 55–80
Gain
Loss
References
8p23.3-q24.3 10p15.3-p12.2 17p11.2-q25.3 2p11.1 4p16.1 8q24.1-q24.3 17q23-q25 8q24.1-q24.2 10p13 17q21-q25.1
9q13-q21.33 10p12.1-q26.3 17p13.2-p11.2 10q24-q25 17p13.3-13.1
(14)
9p21.3 9q21.3 10p11-p15.1 10q23.3-q26 17p12-p13.3 9q22.1-q31 10p11.23-p11.22 17p13.2-p13.1 20q13.2-q13.32
(16)
2p25.2-q14 4p16.2-q13.1 5p15-p13.2 9q31-q34 14q24.2-q32.2 17q12-q24
(15)
BKP1 cell line
61
Letter to the Editor
NFKB pathway is correlated with cell proliferation, cell growth and resistance to apoptosis. Indeed, the activation of this pathway has been shown by Sors et al. (13) in SS. These authors have also demonstrated that selective inhibition of NFKB pathway activation in CTCL cell lines induced cellular apoptosis. Subsequently, they also tested the effect of proteasome inhibitors, which reduce the degradation of the NFKB pathway inhibitors. This causes a decrease in the constitutive activation of NFKB causing cellular apoptosis. This suggests that proteasome inhibitors may, in the future, play a role in the management of patients with SS. Comparison of aCGH data obtained for BKP1 with literature shows common abnormalities (Table 1). Indeed, according to the literature, the signalling pathways or genes involved are TP53, CMYC, NFKB and IL2 (14,15). For BKP1, abnormalities are present in all these genes or signalling pathways except for the gene CMYC. While the existing SS cell lines are characterized in a very global way in particular for the genetic features (i.e. approximate karyotype with suspected chromosomal abnormalities and many
References
1 Willemze R, Kerl H, Sterry W et al. Blood 1997: 90: 354–371. 2 Rook A H, Gottlieb S L, Wolfe J T et al. Clin Exp Immunol 1997: 107(Suppl 1): 16–20. 3 Kaltoft K, Bisballe S, Rasmussen H F et al. Arch Dermatol Res 1987: 279: 293–298. 4 Poszepczynska E, Bagot M, Echchakir H et al. Blood 2000: 96: 1056–1063. 5 Nikolova M, Echchakir H, Wechsler J et al. Br J Dermatol 2003: 148: 24–29. 6 Dummer R, Sigg-Zemann S, Kalthof K et al. Dermatol Basel Switz 1994: 189: 120–124. 7 Litvinov I V, Kupper T S, Sasseville D. Exp Dermatol 2012: 21: 964–966. 8 Ortonne N, Le Gouvello S, Tabak R et al. Exp Dermatol 2012: 21: 461–463. 9 Laharanne E, Chevret E, Idrissi Y et al. Mod Pathol 2010: 23: 547–558.
unidentified chromosomes), BKP1 is exhaustively characterized thanks to the association of conventional karyotype associated with several molecular cytogenetics techniques (FISH and MultiFISH). Using the aCGH, we identified unbalanced gene abnormalities with better resolution than previously described in HUT78, PNO and Se-Ax cell lines (16). We conclude that in addition to the very few SS cell line available, the novel cell line BKP1 has been thoroughly characterized to become a useful tool for basic research and screening of new antilymphoma drugs.
Acknowledgements Prof. Costello and Dr kahn-Perles designed the research. Dr Boudjarane analysed the data and prepared manuscript. Dr Popovici, Dr Zattara, Dr Lafage-Pochitaloff and Dr Boudjarane performed cytogenetic analysis. Prof. Gabert, Dr Essaydi and Dr Beaufils performed molecular analysis (T clonality). Dr Lacroix, Dr Berda, Dr Nicolino-Brunet and Dr Letreut performed flow cytometry and cytology analysis. Dr Farnault collected clinical data.
Conflict of interests The authors have declared no conflicting interests.
10 Zhang C, Toulev A, Kamarashev J et al. Hum Pathol 2007: 38: 995–1002. 11 Barba G, Matteucci C, Girolomoni G et al. Cancer Genet Cytogenet 2008: 184: 48–51. 12 Qin J Z, Kamarashev J, Zhang C L et al. J Invest Dermatol 2001: 117: 583–589. 13 Sors A, Jean-Louis F, Pellet C et al. Blood 2006: 107: 2354–2363. 14 Caprini E, Cristofoletti C, Arcelli D et al. Cancer Res 2009: 69: 8438–8446. 15 Vermeer M H, van Doorn R, Dijkman R et al. Cancer Res 2008: 68: 2689–2698. 16 Lin W M, Lewis J M, Filler R B et al. J Invest Dermatol 2012: 132: 188–197.
Supporting Information
Figure S1. TCRc VJ analysis of BKP1 cell line. Figure S2. Conventional karyotype, multi FISH and specific FISH analysis (from left to right) of the BKP1 cell line. Figure S3. Results of aCGH analysis of BKP1 cell line. Figure S4. Blood cytology of patient from which the BKP1 cell line was obtained. Table S1. Identification of TCRc VJ regions. Table S2. Cytogenetics results obtained by conventional karyotype, multi-FISH and specific FISHs. Table S3. Description of unbalanced chromosomal rearrangements identified by aCGH in the BKP1 cell line. Data S1. Material and methods.
Additional supporting data may be found in the supplementary information of this article.
DOI: 10.1111/exd.12571 www.wileyonlinelibrary.com/journal/EXD
Letter to the Editor
Various peroxisome proliferator-activated receptor (PPAR)-c agonists differently induce differentiation of cultured human keratinocytes Yan Yan1,2, Minao Furumura1, Sanae Numata1, Kwesi Teye1, Tadashi Karashima1, Bungo Ohyama1, Norifumi Tanida3 and Takashi Hashimoto1 1
Department of Dermatology, Kurume University School of Medicine, and Kurume University Institute of Cutaneous Cell Biology, Kurume, Japan; Department of Dermatology, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China; 3Tsukuba Research Laboratories, Hisamitsu Pharmaceutical Co., Inc., Tsukuba, Japan Correspondence: Takashi Hashimoto, MD, Department of Dermatology, Kurume University School of Medicine, and Kurume University Institute of Cutaneous Cell Biology, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan, Tel.: +81-942-31-7853, Fax: +81-942-34-2620, e-mail: [email protected] 2
Abstract: Peroxisome proliferator-activated receptors (PPARs) are potentially useful for the treatment of skin diseases, because they stimulate keratinocyte differentiation, exert anti-inflammatory
62
effects and improve barrier function. We examined five PPAR-c agonists, including four thiazolidinediones (ciglitazone, troglitazone, rosiglitazone and pioglitazone) and an angiotensin-II
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 55–80
7 Poeggeler B, Schulz C, Pappolla M A et al. Exp Dermatol 2010: 19: 12–18. 8 Sumikawa Y, Inui S, Nakajima T et al. Exp Dermatol 2014: 23: 27–32. 9 Ibrahim L, Wright E A. J Embryol Exp Morphol 1975: 33: 831–844. 10 Van Harmelen V, Reynisdottir S, Eriksson P et al. Diabetes 1998: 47: 913–917.
Supporting Information Additional supporting data may be found in the supplementary information of this article. Figure S1. A cross section of mouse dorsal skin is showing the dermal white adipose tissue (DWAT) below dermis. Figure S2. The levels of leptin, adiponectin, BMP-2, and BMP-4 mRNA expressed by adipocytes and preadipocytes were measured with QPCR. Preadipocytes were isolated from DWAT of 5-week-old female C56BL/6 mice.
Figure S3. The longitudinal sections of anagen vibrissa hair follicles demonstrate where DWAT surrounds the hair follicle. Figure S4. (a) A schematic representation of expression patterns of adipose-derived factors throughout hair cycle stages from hair follicles and dermal adipose tissue. Dashed lines are hypothetical (yet to be determined). ‘C’: catagen; ‘EA’: early anagen; ‘ET’: early telogen; ‘LA’: late anagen; ‘LT’: late telogen. (b) A diagrammatic representation of the results collective from the literature showing the effects of paracrines, i.e., leptin, adiponectin, BMP-2, BMP-4, and PDGF, from skin adipocytes or preadipocytes on hair follicle growth. ‘?’: stimulatory; ‘┤’: inhibitory. Dashed lines are ‘hypothetical’. In telogen, leptin from adipocytes (reference 1) and PDGF from preadipocytes (reference 2) serve as anagen inducers by activating hair follicle epithelial stem cells and, therefore, hair growth. In contrast, BMP-2 and BMP-4 from adipocytes inhibits stem
cell activation and, therefore, blocking anagen induction (reference 3). The size of dermal adipose tissue is largely reduced in telogen, which suggests that a regulatory feedback loop from the hair follicle might exist in regulating adipose tissue metabolism. In anagen, leptin from adipocytes may contribute to the inhibitory effect which the adipocytes have on hair growth through possibly leptin receptors on the epithelial stem cells (this study). Although with mechanism unknown, the preadipocytes also exhibit an inhibitory effect on hair growth on anagen phase hair follicle (this study). On the other hand, adiponectin (derived from adipocytes) seems to promote hair growth at this stage (reference 4). The size of dermal adipose tissue increases during this time indicating that a positive signal might exist from the hair follicle to stimulate the expansion of the adipose tissue. Data S1. Material and methods. Data S2. Acknowledgements.
DOI: 10.1111/exd.12567
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
Characterization of the novel Sezary lymphoma cell line BKP1 John Boudjarane1,2, Arnaud Essaydi3, Laure Farnault2,4, Cornel Popovici1, Marina Lafage-Pochitaloff1,2, €l Berda-Haddad5, Romaric Lacroix5, Corinne Nicolino-Brunet5, The re se Le Treut6, Nathalie Beaufils3, Yae 1,2 2,3,7 2,8 2,4,8 s and Re gis Costello le ne Zattara , Jean Gabert , Brigitte Kahn-Perle He 1 Departement de Genetique Medicale, H^ opital La Timone, Assistance Publique-H^ opitaux de Marseille, Marseille, France; 2Aix-Marseille Universite, opital Nord, Marseille, France; 4Service d’Hematologie, H^ opital La Marseille, France; 3Laboratoire de Biochimie et de Biologie Moleculaire, H^ Conception, Marseille, France; 5Laboratoire d’Hematologie, H^ opital La Conception, Marseille, France; 6Laboratoire d’Hematologie, H^ opital Nord, Marseille, France; 7Unite INSERM 1072, Marseille, France; 8Technologies Avancees pour la Genomique et la Clinique (TAGC)/unite INSERM U1090, Marseille, France Correspondence: Regis T Costello, Service d’Hematologie, Centre Hospitalier Universitaire La Conception, 147 Boulevard Baille, 13005 Marseille, France, Tel.: +(33)-4-91-38-41-50, Fax: +(33)-4-91-38-41-54, e-mail: [email protected]
Abstract: Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of lymphomas primarily involving the skin. The most common types are mycosis fungoides (MF) and Sezary Syndrome (SS). We report a novel long-term fast-growing SS line termed BKP1 that was characterized by flow cytometry (FC), conventional and molecular cytogenetic [FISH/multi-FISH together with array comparative genomic hybridization (aCGH)]. FC immunophenotype of the BKP1 is CD2+CD5+CD3+ CD4+CD8 CD7 CD25 CD26 CD30 CD158k+. The TCRc characterization of BKP1 by PCR identified a clonal rearrangement. The conventional cytogenetic and Multi-FISH analysis showed complex chromosomal rearrangements. aCGH
analysis highlighted the loss of genes involved in cell cycle control, in immune response (HLA, complement complex) and DNA damage repair mechanisms. The BKP1 is another lymphoma cell line thoroughly characterized that can be a valuable tool for both basic and applied research such as identification of deregulated genes and/or pathways and screening for new antilymphoma drugs.
Introduction
only four SS cell lines [Se-Ax (3), HUT78, Pno (4) and Cou (5)] and one MF cell line [MyLA 2059 (6)] have been published.
Cutaneous T-cell lymphomas are a heterogeneous group primarily involving the skin (1). Most common types are MF and SS that are characterized by invasion of skin, lymph nodes or blood (SS) by CD4+ T cells phenotypically close to memory-effector T cells for MF and central-memory T cells for SS (2). Treatment of SS is disappointing even though a large number of drugs have been tested in clinical trials. The possibility to test in vitro and in vivo (animal models) SS cells is an important issue to identify novel drugs. So far,
60
Key words: cutaneous lymphoma – karyotype – T-cell lymphoma
Accepted for publication 10 October 2014
Questions addressed We report a novel long-term fast-growing SS line termed BKP1 that was characterized by FC phenotype, conventional karyotype completed by fluorescent in situ hybridization (FISH)/multi-FISH study together with aCGH study. This study represents the most comprehensive characterization, to our knowledge, of a novel SS cell line.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 55–80
Letter to the Editor
Experimental design Peripheral blood from SS patient was obtained after informed consent. Mononucleated cells were isolated by Ficoll-Paque density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) and cultured in 24-well culture plate in RPMI medium (BioWhittaker, Verviers, Belgium) containing 10% foetal calf serum (FCS, Perbio Science, Brebieres, France), interleukin (IL)-7 10 ng/ml (Genzyme, Cambridge, UK) and anti-CD28 monoclonal antibody 248 (D Olive, 1/400 dilution) at a 106 cells/ml initial concentration.
Results Flow cytometry immunophenotyping of both patient’s PB at diagnosis and BKP1 (three phenotypic assays after 1, 3 and 6 months culture) showed the same expression profile: CD2+CD5+CD3+CD4+CD8 CD7 CD25 CD26 CD30 CD158k+ (Fig. 1). FC Vb repertoire analyses were performed on patient’s PB at diagnosis and on the BKP1 (two phenotypic assays after 1 and 6 months culture). FC Vb repertoire indicates the clonal expansion of T cells (Fig. 1). Analysis of PCR TCR c VJ product showed the two clonal products (one in each tube A and B) in both patient’s PB at diagnosis, BKP1 (6 months culture; Figure S1) and skin biopsy with the same following sizes: tube A, an unique fragment of 257 base pairs, tube B, an unique fragment of 190 base pairs. Sequencing of the PCR products and in silico analysis using IMGT database allowed the identification of the TCRc VJ rearrangement (Table S1).
(a)
The conventional R-bands cytogenetic analysis of cell line showed a complex karyotype with numerical and structural abnormalities that was completed by multi-FISH analysis (Figure S2 and Table S2). Comprehensive genomic profile of gains and losses for BKP1 was generated with aCGH (Figure S3 and Table S3). The aCGH profile of BKP1 confirmed the complex patterns of rearrangements previously demonstrated with multi-FISH and conventional cytogenetics. We do not observe any anomaly in 6q23.3 and 11p15.5 regions. These two regions contain respectively AHI1 and CDKN1C genes, described to have a role in CTCL progression (7).
Discussion We show that BKP1 expresses compatible immunophenotype with Sezary cells. Indeed, we found CD158k expression which is recurrently expressed by neoplastic cells in SS (8) and CD30 and CD26 are negative. We identified with the aCGH genetic defects on genes involved in carcinogenesis of MF and SS. Firstly, we have detected the deletion of CDKN2A (p16) and CDKN2B (p15) genes, leading to a loss of cell cycle control. This anomaly is frequently observed in the late stages of CTCL and is associated with poor prognosis (9). Zhang et al. (10) showed a decreased expression of p16 in the skin lesions of MF and SS late stage evoking a probable proliferative advantage to cells having lost the expression of p16. We also identified in BKP1 cell line a deletion of FANCC, FANCM and XPA. These three genes are involved in the repair of DNA damage so that their deletions could lead to genomic instability. We observed in the BKP1 cell line a duplication 17q that is a recurrent (17–28%) abnormality considered as an early pathogenic event that is involved in SS cell proliferation advantage (11). The 17q region includes STAT3 and STAT5 genes, which are transcription factors involved in oncogenesis since constitutively activated in CTCL (12). These transcription factors activate the Ras/MEK/ERK and PI3K/Akt pathways, suggesting clues for the use of metabolic inhibitors for treatment of MF and SS. BKP1 also presents a gain of REL gene (2p13) and loss of IKBA (14q13). Both genes are involved in the NFKB pathway, REL as an activator and IKBA as an inhibitor. The gain of an activator and loss of an inhibitor of NFKB signalling pathway could contribute to the constitutive activation of this pathway. Constitutive activation of
(b) Table 1. Comparison of BKP1 cell line aCGH abnormalities with those of the literature
Figure 1. Flow cytometry analysis of BKP1 cell line. (a) Analysis of the TcR Vbeta repertoire by flow cytometry. The Sezary cells were analysed using a TcR Vbeta panel covering 70% of the TcR Vbeta repertoire. In each quadrant is indicated the Vbeta reactivity tested. (b) Flow cytometry analysis of KIRDL2/CD158k, DDIV/CD26 (R&D Systems Minneapolis Europe) and CD30 (Beckman Coulter). There are singleparameter histograms with: 1 On ● ● ●
the left: Isotypic PE control (purple) with a mean fluorescence intensity (MFI) of 0,13 CD26 (green) with MFI of 0,20 CD158k (blue) with MFI of 0,83
2 On the right: ● CD30 (red) with MFI 0,10.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 55–80
Gain
Loss
References
8p23.3-q24.3 10p15.3-p12.2 17p11.2-q25.3 2p11.1 4p16.1 8q24.1-q24.3 17q23-q25 8q24.1-q24.2 10p13 17q21-q25.1
9q13-q21.33 10p12.1-q26.3 17p13.2-p11.2 10q24-q25 17p13.3-13.1
(14)
9p21.3 9q21.3 10p11-p15.1 10q23.3-q26 17p12-p13.3 9q22.1-q31 10p11.23-p11.22 17p13.2-p13.1 20q13.2-q13.32
(16)
2p25.2-q14 4p16.2-q13.1 5p15-p13.2 9q31-q34 14q24.2-q32.2 17q12-q24
(15)
BKP1 cell line
61
Letter to the Editor
NFKB pathway is correlated with cell proliferation, cell growth and resistance to apoptosis. Indeed, the activation of this pathway has been shown by Sors et al. (13) in SS. These authors have also demonstrated that selective inhibition of NFKB pathway activation in CTCL cell lines induced cellular apoptosis. Subsequently, they also tested the effect of proteasome inhibitors, which reduce the degradation of the NFKB pathway inhibitors. This causes a decrease in the constitutive activation of NFKB causing cellular apoptosis. This suggests that proteasome inhibitors may, in the future, play a role in the management of patients with SS. Comparison of aCGH data obtained for BKP1 with literature shows common abnormalities (Table 1). Indeed, according to the literature, the signalling pathways or genes involved are TP53, CMYC, NFKB and IL2 (14,15). For BKP1, abnormalities are present in all these genes or signalling pathways except for the gene CMYC. While the existing SS cell lines are characterized in a very global way in particular for the genetic features (i.e. approximate karyotype with suspected chromosomal abnormalities and many
References
1 Willemze R, Kerl H, Sterry W et al. Blood 1997: 90: 354–371. 2 Rook A H, Gottlieb S L, Wolfe J T et al. Clin Exp Immunol 1997: 107(Suppl 1): 16–20. 3 Kaltoft K, Bisballe S, Rasmussen H F et al. Arch Dermatol Res 1987: 279: 293–298. 4 Poszepczynska E, Bagot M, Echchakir H et al. Blood 2000: 96: 1056–1063. 5 Nikolova M, Echchakir H, Wechsler J et al. Br J Dermatol 2003: 148: 24–29. 6 Dummer R, Sigg-Zemann S, Kalthof K et al. Dermatol Basel Switz 1994: 189: 120–124. 7 Litvinov I V, Kupper T S, Sasseville D. Exp Dermatol 2012: 21: 964–966. 8 Ortonne N, Le Gouvello S, Tabak R et al. Exp Dermatol 2012: 21: 461–463. 9 Laharanne E, Chevret E, Idrissi Y et al. Mod Pathol 2010: 23: 547–558.
unidentified chromosomes), BKP1 is exhaustively characterized thanks to the association of conventional karyotype associated with several molecular cytogenetics techniques (FISH and MultiFISH). Using the aCGH, we identified unbalanced gene abnormalities with better resolution than previously described in HUT78, PNO and Se-Ax cell lines (16). We conclude that in addition to the very few SS cell line available, the novel cell line BKP1 has been thoroughly characterized to become a useful tool for basic research and screening of new antilymphoma drugs.
Acknowledgements Prof. Costello and Dr kahn-Perles designed the research. Dr Boudjarane analysed the data and prepared manuscript. Dr Popovici, Dr Zattara, Dr Lafage-Pochitaloff and Dr Boudjarane performed cytogenetic analysis. Prof. Gabert, Dr Essaydi and Dr Beaufils performed molecular analysis (T clonality). Dr Lacroix, Dr Berda, Dr Nicolino-Brunet and Dr Letreut performed flow cytometry and cytology analysis. Dr Farnault collected clinical data.
Conflict of interests The authors have declared no conflicting interests.
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Supporting Information
Figure S1. TCRc VJ analysis of BKP1 cell line. Figure S2. Conventional karyotype, multi FISH and specific FISH analysis (from left to right) of the BKP1 cell line. Figure S3. Results of aCGH analysis of BKP1 cell line. Figure S4. Blood cytology of patient from which the BKP1 cell line was obtained. Table S1. Identification of TCRc VJ regions. Table S2. Cytogenetics results obtained by conventional karyotype, multi-FISH and specific FISHs. Table S3. Description of unbalanced chromosomal rearrangements identified by aCGH in the BKP1 cell line. Data S1. Material and methods.
Additional supporting data may be found in the supplementary information of this article.
DOI: 10.1111/exd.12571 www.wileyonlinelibrary.com/journal/EXD
Letter to the Editor
Various peroxisome proliferator-activated receptor (PPAR)-c agonists differently induce differentiation of cultured human keratinocytes Yan Yan1,2, Minao Furumura1, Sanae Numata1, Kwesi Teye1, Tadashi Karashima1, Bungo Ohyama1, Norifumi Tanida3 and Takashi Hashimoto1 1
Department of Dermatology, Kurume University School of Medicine, and Kurume University Institute of Cutaneous Cell Biology, Kurume, Japan; Department of Dermatology, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China; 3Tsukuba Research Laboratories, Hisamitsu Pharmaceutical Co., Inc., Tsukuba, Japan Correspondence: Takashi Hashimoto, MD, Department of Dermatology, Kurume University School of Medicine, and Kurume University Institute of Cutaneous Cell Biology, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan, Tel.: +81-942-31-7853, Fax: +81-942-34-2620, e-mail: [email protected] 2
Abstract: Peroxisome proliferator-activated receptors (PPARs) are potentially useful for the treatment of skin diseases, because they stimulate keratinocyte differentiation, exert anti-inflammatory
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effects and improve barrier function. We examined five PPAR-c agonists, including four thiazolidinediones (ciglitazone, troglitazone, rosiglitazone and pioglitazone) and an angiotensin-II
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 55–80
