窖蛋白一(caveolin-1)是一種骨架蛋白，可以調節細胞膜窖構造的形成，並與細胞膜窖構造上附著的訊息分子作用而影響細胞反應。胃癌細胞內窖蛋白一含量的改變與細胞癌化有關。此外，致癌因子所誘發的細胞轉形也會減少酪氨酸磷酸化的窖蛋白一。為了進一步研究窖蛋白一的表現及其磷酸化與細胞癌化之間的關係，窖蛋白一與十四號酪氨酸突變為苯胺基丙酸的磷酸化缺陷之窖蛋白一(caveolin-1/Y14F，以下簡稱磷酸化缺陷窖蛋白一)之表達基因被送入一種不會表達窖蛋白一的胃癌細胞AGS中。在此，圓點墨點法被選擇來做快速、大量、容易使用且不複雜的篩選及定量於AGS轉形細胞內的窖蛋白一及磷酸化缺陷窖蛋白一。由圓點墨點法的結果顯示在AGS轉形細胞，十一株中的九株有表達窖蛋白一，另外十一株中的七株有表達磷酸化缺陷窖蛋白一。此外，關於定量AGS轉形細胞內窖蛋白一的表現上，圓點墨點法所得的結果與西方墨點法相似。然而，在定量磷酸化缺陷窖蛋白一時，圓點墨點法所得結果約比西方墨點法高五倍。在定量磷酸化缺陷窖蛋白一的結果上的不同是因為窖蛋白一抗體對磷酸化缺陷窖蛋白一的親和性較低。雖然在定量AGS轉形細胞內表達的磷酸化缺陷窖蛋白一的含量仍須進一步研究，圓點墨點法在AGS轉形細胞篩選及定量窖蛋白一的表達量時可以作為一個有效的工具。圓點墨點法應該也可以用於篩選其他細胞株內窖蛋白一的表達或減少表達。

Caveolin-1 is a scaffolding protein that mediates caveolae formation and gives rise to cellular responses by interacting with caveolae-localized signal molecules. The various level of caveolin-1 expressed in gastric cancer (GC) cells is associated with oncogenesis. Besides, the oncogene-induced cell transformation reduces the tyrosine phosphorylation status of caveolin-1. To further investigate the association of expression of caveolin-1 and its phosphorylation status with oncogenesis, caveolin-1 and phosphorylation-deficient caveolin-1/Y14F mutant were introduced into gastric cancer AGS cells. Here, the dot blot analysis was chosen to achieve time-saving, large-scale, generic-to-use, and low-complexity in screening and quantification of the expression of caveolin-1 and caveolin-1/Y14F in the collection of AGS transfectants. After analysis by dot blot, 9 out of 11 and 7 out of 11 of AGS transfectants stably express caveolin-1 and caveolin-1/Y14F, respectively. Moreover, with respect to the quantification of caveolin-1 expression in AGS transfectants, the dot blot analysis showed results similar to those yielded by the western blot analysis. However, the dot blot analysis showed about 5-fold higher than western blot analysis in quantification of the amount of caveolin-1/Y14F. The differences in the quantification of caveolin-1/Y14F between the dot blot and western blot analyses were due to the decrease in binding affinity of anti-caveolin-1 antibody to the caveolin-1/Y14F. Although the amount of caveolin-1/Y14F expressed in AGS transfectants is needed for further investigation, dot blot analysis can serve as an efficient tool for screening and quantification of the amount of caveolin-1 expressed in AGS transfectants. Dot blot should also be able to be applied in screening the expression or knockdown of caveolin-1 in other cell lines.

References

Anderson, H. A., Chen, Y., and Norkin, L. C. (1996). Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae. Mol. Biol. Cell 7, 1825-1834.
Burgermeister, E., Xing, X., Rocken, C., Juhasz, M., Chen, J., Hiber, M., Mair, K., Shatz, M., Liscovitch, M., Schmid, R. M., and Ebert, M. P. (2007). Differential expression and function of caveolin-1 in human gastric cancer progression. Cancer Res. 67, 8519-8526.
Campbell, S. M., Crowe, S. M., and Mak, J. (2001). Lipid rafts and HIV-1: from viral entry to assembly of progeny virions. J. Clin. Virol. 22, 217-227.
Cao, H., Courchesne, W. E., and Mastick, C. C. (2002). A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase. J. Biol. Chem. 277, 8771-8774.
Capozza, F., Williams, T. M., Schubert, W., McClain, S., Bouzahzah, B., Sotgia, F., and Lisanti, M. P. (2003). Absence of caveolin-1 sensitizes mouse skin to carcinogen-induced epidermal hyperplasia and tumor formation. Am. J. Pathol. 162, 2029-2039.
del Pozo, M. A., Balasubramanian, N., Alderson, N. B., Kiosses, W. B., Grande-Garcia, A., Anderson, R. G., and Schwartz, M. A. (2005). Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization. Nat. Cell Biol. 7, 901-908.
Engelman, J. A., Wykoff, C. C., Yasuhara, S., Song, K. S., Okamoto, T., and Lisanti, M. P. (1997). Recombinant expression of caveolin-1 in oncogenically transformed cells abrogates anchorage-independent growth. J. Biol. Chem. 272, 16374-16381.
Engelman, J. A., Zhang, X. L., and Lisanti, M. P. (1998). Genes encoding human caveolin-1 and -2 are co-localized to the D7S522 locus (7q31.1), a known fragile site (FRA7G) that is frequently deleted in human cancers. FEBS Lett. 436, 403-410.
Engelman, J. A., Zhang, X. L., and Lisanti, M. P. (1999). Sequence and detailed organization of the human caveolin-1 and -2 genes located near the D7S522 locus (7q31.1). Methylation of a CpG island in the 5' promoter region of the caveolin-1 gene in human breast cancer cell lines. FEBS Lett. 448, 221-230.
Fielding, C. J. and Fielding,P.E. (1997). Intracellular cholesterol transport. J. Lipid Res. 38, 1503-1521.
Glenney, J. R., Jr. (1989). Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus. J. Biol. Chem. 264, 20163-20166.
Hayashi, K., Matsuda, S., Machida, K., Yamamoto, T., Fukuda, Y., Nimura, Y., Hayakawa, T., and Hamaguchi, M. (2001). Invasion activating caveolin-1 mutation in human scirrhous breast cancers. Cancer Res. 61, 2361-2364.
Ikonen, E. and Parton, R. G. (2000). Caveolins and cellular cholesterol balance. Traffic. 1, 212-217.
Koleske, A. J., Baltimore, D., and Lisanti, M. P. (1995). Reduction of caveolin and caveolae in oncogenically transformed cells. Proc. Natl. Acad. Sci. U. S. A 92, 1381-1385.
Kurzchalia, T. V., Dupree, P., Parton, R. G., Kellner, R., Virta, H., Lehnert, M., and Simons, K. (1992). VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. J. Cell Biol. 118, 1003-1014.
Lee, S. W., Reimer, C. L., Oh, P., Campbell, D. B., and Schnitzer, J. E. (1998). Tumor cell growth inhibition by caveolin re-expression in human breast cancer cells. Oncogene 16, 1391-1397.
Li, S., Seitz, R., and Lisanti, M. P. (1996). Phosphorylation of caveolin by src tyrosine kinases. The alpha-isoform of caveolin is selectively phosphorylated by v-Src in vivo. J. Biol. Chem. 271, 3863-3868.
Liu, P., Rudick, M., and Anderson, R. G. (2002). Multiple functions of caveolin-1. J. Biol. Chem. 277, 41295-41298.
Lu, T. L., Kuo, F. T., Lu, T. J., Hsu, C. Y., and Fu, H. W. (2006). Negative regulation of protease-activated receptor 1-induced Src kinase activity by the association of phosphocaveolin-1 with Csk. Cell Signal. 18, 1977-1987.
Norkin, L. C. (2001). Caveolae in the uptake and targeting of infectious agents and secreted toxins. Adv. Drug Deliv. Rev. 49, 301-315.
Okamoto, T., Schlegel, A., Scherer, P. E., and Lisanti, M. P. (1998). Caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J. Biol. Chem. 273, 5419-5422.
Pelkmans, L. and Helenius, A. (2002). Endocytosis via caveolae. Traffic. 3, 311-320.
Schwartz, M. A. (1997). Integrins, oncogenes, and anchorage independence. J. Cell Biol. 139, 575-578.
Simons, K. and Toomre, D. (2000). Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31-39.
Smart, E. J., Graf, G. A., McNiven, M. A., Sessa, W. C., Engelman, J. A., Scherer, P. E., Okamoto, T., and Lisanti, M. P. (1999). Caveolins, liquid-ordered domains, and signal transduction. Mol. Cell Biol. 19, 7289-7304.
Thompson, T. C., Timme, T. L., Li, L., and Goltsov, A. (1999). Caveolin-1, a metastasis-related gene that promotes cell survival in prostate cancer. Apoptosis. 4, 233-237.
Williams, T. M., Cheung, M. W., Park, D. S., Razani, B., Cohen, A. W., Muller, W. J., Di, V. D., Chopra, N. G., Pestell, R. G., and Lisanti, M. P. (2003). Loss of caveolin-1 gene expression accelerates the development of dysplastic mammary lesions in tumor-prone transgenic mice. Mol. Biol. Cell 14, 1027-1042.
Williams, T. M. and Lisanti, M. P. (2004). The caveolin proteins. Genome Biol. 5, 214.
Yang, G., Timme, T. L., Frolov, A., Wheeler, T. M., and Thompson, T. C. (2005). Combined c-Myc and caveolin-1 expression in human prostate carcinoma predicts prostate carcinoma progression. Cancer 103, 1186-1194.