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Differential expression of CD44(S) and variant isoforms v3, v10 in three-dimensional cultures of mouse melanoma cell lines

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Abstract

Multi-cellular spheroids (MCS) generated from tumor cells serve as excellent in vitro models for understanding the mechanisms of tumor progression and micro-metastasis. We have compared the expression of molecular markers with reference to their growth as conventional adherent monolayers (2-D) and anchorage independent cultures (3-D) using two mouse melanoma cell lines, B16F10 and Clone M3. The two cell lines differed in their ability to form spheroids with respect to their aggregation potential, with B16F10 forming large clusters compared to Clone M3. A panel of molecular markers comprising cell adhesion molecules, cyclin dependent kinase inhibitors and members of the cadherin–catenin complex were analyzed by flow cytometry in 2-D and 3-D cultures. There was a distinct difference in the patterns of expression of CD44(S) and variant isoforms v3,v10 in spheroids compared to cells grown as monolayers in both cell lines. Also, there was an increase in cells positive for CDK inhibitor p27 in 3-D cultures from the B16F10 cell line. The expression of alpha and gamma catenin was down regulated in spheroids. As these molecules are implicated in the regulation of cell proliferation, alterations in the expression of these molecules in 3-D cultures compared to their 2-D counterparts suggests the importance of spheroids as experimental model for tumorigenesis.

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References

  1. La Rue KE, Bradbury EM, Freyer JP. Differential regulation of cyclin-dependent kinase inhibitors in monolayer and spheroid cultures of tumorigenic and nontumorigenic fibroblasts. Cancer Res 1998; 58(6): 1305–14.

    CAS  Google Scholar 

  2. Hamilton G. Multi-cellular spheroids as an in vitro tumor model. Cancer Lett 1998; 131(1): 29–34.

    Article  PubMed  CAS  Google Scholar 

  3. Santini MT, Rainaldi G. Three dimensional spheroid model in tumor biology Pathobiology 1999; 67: 148–57.

    Article  PubMed  CAS  Google Scholar 

  4. Rak J, Mitsuhashi Y, Erdos V et al. Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression. J Cell Biol 1995; 131(6): 1587–98.

    Article  PubMed  CAS  Google Scholar 

  5. Desoize B, Gimonet D, Jawdiller JC. Cell Culture as spheroids: An approach to multi-cellular resistance. Anticancer Res 1988; 18(6A): 4147–58.

    Google Scholar 

  6. Bates RC, Edwards NS, Yates JD. Spheroids and cell survival. Crit Rev Oncol Hematol 2000; 36(2–3): 61–74.

    PubMed  CAS  Google Scholar 

  7. Rafstad EK, Wahl A, Davies C et al. Growth characteristics of human melanoma multi-cellular spheroids in liquid-overlay culture: Comparisons with the parent tumor xenografts. Cell Tissue Kinet 1986; 19: 205–16.

    Google Scholar 

  8. Mueller-Klieser W. Three-dimensional cell cultures: From molecular mechanisms to clinical applications. Am J Pathol 1997; 273(4): C1109–23.

    CAS  Google Scholar 

  9. Lawlor ER, Schcel C, Irving J et al. Anchorage — independent multicellular spheroids as an in vitro model of growth signaling in Ewing tumors. Oncogene 2002; 21: 307–18.

    Article  PubMed  CAS  Google Scholar 

  10. Bartolazzi A, Peach R, Aruffo A et al. Interaction between CD44 and hyaluronate is directly implicated in the regulation of tumor development. J Exp Med 1994; 180: 53–66.

    Article  PubMed  CAS  Google Scholar 

  11. Tolg C, Hofmann M, Herrlich P et al. Splicing choice from ten variant exons establishes CD44 variability. Nucleic Acids Res 1993; 21: 1225–9.

    PubMed  CAS  Google Scholar 

  12. Screaton GR, Bell MV, Jackson DG et al. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci USA 1992; 89: 12160–4.

    Article  PubMed  CAS  Google Scholar 

  13. Koopman G, Hieder KH, Horst E et al. Activated human lymphocytes and aggressive non-Hodgkin's lymphomas express a homologue of the metastasis associated variant of CD44. J Exp Med 1993; 177: 879-904.

    Article  Google Scholar 

  14. Wielenga VJM, Heider KH, Offerhans JA et al. Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression. Cancer Res 1993; 53: 4754–6.

    PubMed  CAS  Google Scholar 

  15. Kaufmann M, Heider KH, Sinn HP et al. CD44 variant exon epitopes in primary breast cancer and length of survival. Lancet 1995; 345: 615–9.

    Article  PubMed  CAS  Google Scholar 

  16. Salmi M, Gron-Virta K, Sointu P et al. Regulated expression of exon v6 containing isoforms of cd44 in man: Down regulation during malignant transformation of tumors of squamo-cellular origin. J Cell Biol 1993; 122: 431-42.

    Article  PubMed  CAS  Google Scholar 

  17. Liu AY, Expression of CD44 in prostate cancer cells. Cancer Lett 1994; 76: 63–9.

    Article  PubMed  Google Scholar 

  18. Seiter S, Schadendorf D, Harrmann K et al. Expression of CD44 variant isoforms in malignant melanoma. Clin Cancer Res 1996; 2: 447–56.

    PubMed  CAS  Google Scholar 

  19. Manten-Horst E, Danen EH, Smit L et al. Expression of CD44 splice variants in human cutaneous melanoma and melanoma cell lines is related to tumor progression and metastatic potential. Int J Cancer 1995; 22(64): 182–8.

    Google Scholar 

  20. Zhu Z, Sanchez-Sweatman O, Huang X et al. Anoikis and metastatic potential of Cloudman S91 melanoma cells. Cancer Res 2001; 61: 1707–16.

    PubMed  CAS  Google Scholar 

  21. Coucke P, De leval L, Leyh P et al. Influence of laminin or fibroblasts upon colony formation in mouse by B16 melanoma cell spheroids: A morphometric analyses. In vivo 1992; 6(2): 119–24.

    PubMed  CAS  Google Scholar 

  22. Raz A, Ben-ze'ev A. Modulation of the metastatic capability in B16 melanoma by cell shape. Science 1983; 221(4617): 1307–10.

    PubMed  CAS  Google Scholar 

  23. StamenKovic I, Aruffok A, Amiot M et al. The hematopoietic and epithelial forms of CD44 are distinet polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J 1991; 10(2): 343–8.

    PubMed  CAS  Google Scholar 

  24. Grimme HU, Termeer CC, Bennett KL et al. Colocalization of basic fibroblast growth factor and CD44 isoforms containing the variably spliced exon v3 (CD44v3) in normal skin and in epidermal skin cancers. Br J Dermatol 1999; 141(5): 824–32.

    Article  PubMed  CAS  Google Scholar 

  25. De Pauw-Gillet MC, Christiane Y, Foidarf JM et al. The mouse B16 melanoma in a 3-dimensional culture. Bull Assoc Anat (Nancy) 1986; 70(210): 21–4.

    CAS  Google Scholar 

  26. Perl AK, Wigenbus P, Dahl U et al. A causal role for E-cadherin in transition from adenoma to carcinoma. Nature 1998; 392: 190–3.

    Article  PubMed  CAS  Google Scholar 

  27. Vieminckx K, Vakaet L, Mareel M et al. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell 1991; 66(1): 107–19.

    Article  Google Scholar 

  28. Dorudi S, Shieffield JP, Poulsom R et al. E.Cadherin expression in colorectal cancer. An immunocytochemical and in situ hybridization study. Am J Pathol 1993; 142: 981–6.

    PubMed  CAS  Google Scholar 

  29. Berx G, Cleton-Jansen AM, Strumane K et al. E.Cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extra-cellular domain. Oncogene 1996; 13: 1919-25.

    PubMed  CAS  Google Scholar 

  30. Graff RJ, Gabrielson E, Fujii H et al. Methylation patterns of the cadherin 5' Cp9 island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression. J Biol Chem 2000; 275(4): 2727-32.

    Article  PubMed  CAS  Google Scholar 

  31. Pierceall WE, Woodard AS, Morrow JS et al. Frequent alterations in E.cadherin and alpha and beta catenin expression in human breast cancer. Oncogene 1995; 11(7): 1319–26.

    PubMed  CAS  Google Scholar 

  32. Von Schilippe M, Marshell JF, Perry P et al. Functional Interaction between E-cadherin and αv containing integrin in carcinoma cells. J Cell Sci 2000; 113: 425–37.

    Google Scholar 

  33. Silye R, Karyiannakis AJ, Syrigos KN et al. E-cadherin/catenin complex in benign and malignant melanocytic lesions. J Pathol 1998; 186(4): 350–5.

    Article  PubMed  CAS  Google Scholar 

  34. Zhain XD, Hersey P. Expression of catenins and p120 cas in melanocytie nevi and cutaneous melanoma: Deficient α-catenin expression is associated with melanoma progression. Pathology 1999; 31(3): 239–46.

    Article  Google Scholar 

  35. Behren J, Von Kries JP, Kuhl M et al. Functional interaction of β-catenin with the transcription factor LEF-2. Nature 1996; 382(6592): 638–42.

    Article  Google Scholar 

  36. Molenaar M, Van de Weterin M, Oosterwegel M et al. XTCf-3 transcription factor mediates β-catenin induced axis formation in xenopus embryos. Cell 1996; 86(3): 391–9.

    Article  PubMed  CAS  Google Scholar 

  37. Rubinfeld B, Robbins P, EI-Gamil M et al. Stabilization of β-catenin by genetic defects in melanoma cell lines. Science 1997; 275: 1790–2.

    Article  PubMed  CAS  Google Scholar 

  38. Hunter T, Pines J. Cyclins and cancer II: Cyclin D and CDK inhibitors come of age. Cell 1994; 79: 573–82.

    Article  PubMed  CAS  Google Scholar 

  39. Morgan DO. Principles of CDK regulation. Nature (London) 1995; 374: 131–4.

    Article  CAS  Google Scholar 

  40. Croix BS, Florenes VA, Rak JW et al. Impact of the cyclin-dependent kinase inhibitor p27 kip1 on resistance of tumor cells to anticancer agents. Nature Med 1996; 2: 1204–10.

    Article  Google Scholar 

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Authors and Affiliations

  1. National Centre for Cell Science (NCCS), NCCS Complex, Ganeshkhind, Pune, 411007, India

    Anjali Shiras, Arti Bhosale, Anjali Patekar, Varsha Shepal & Padma Shastry

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  1. Anjali Shiras
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  2. Arti Bhosale
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  3. Anjali Patekar
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  4. Varsha Shepal
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  5. Padma Shastry
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Shiras, A., Bhosale, A., Patekar, A. et al. Differential expression of CD44(S) and variant isoforms v3, v10 in three-dimensional cultures of mouse melanoma cell lines. Clin Exp Metastasis 19, 445–455 (2002). https://doi.org/10.1023/A:1016305611858

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