A role of fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy, in cancer cells: a possible role to suppress cell proliferation

Abstract

Fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy (FCMD), is presumably related to the glycosylation of alpha-dystroglycan (alpha-DG), involved in basement membrane formation. Hypoglycosylation of alpha-DG plays a key role for the pathogenesis of FCMD. On the other hand, fukutin and alpha-DG are also expressed in various non-neuromuscular tissues. Recently, a role of alpha-DG as a cancer suppressor has been proposed, because of a decrease of glycosylated alpha-DG in cancers. In this study, function of fukutin was investigated in two cancer cell lines, focusing on whether fukutin is involved in the glycosylation of alpha-DG in cancer cells and has any possible roles related to a cancer suppressor. Localization of fukutin and a result of laminin-binding assay after RNA interference suggest that fukutin may be involved in the glycosylation of alpha-DG in a small portion in these cancer cell lines. In Western blotting and immuno-electron microscopy, localization of fukutin in the nucleus was suggested in addition to the Golgi apparatus and/or endoplasmic reticulum. Immunohistochemically, there were more Ki-67-positive cells and more nuclear staining of phosphorylated c-jun after knockdown of fukutin in two cell lines. Fukutin appears to suppress cell proliferation through a system involving c-jun, although it is unclear this process is related to alpha-DG or not at present. The result may propose a possibility of another function of fukutin in addition to the glycosylation of alpha-DG in cancer cells.

Figure 1

Dystrophin-glycoprotein complex in striated muscle.

Figure 2

Western blotting for fukutin in control tissues and HeLa cells (a, b). A band at about 60 kDa is seen in total extracts of skin, pancreas, kidney, uterine cervix and HeLa cells (a). Both nuclear and cytoplasmic extracts of HeLa cells exhibit a band at about 60 kDa (arrow), and nuclear extracts show stronger reaction compared with cytoplastic extracts (b). There are no bands in negative controls. Sk, skin; Pn, pancreas; Kd, kidney; Cx, uterine cervix; NC, negative control; Cy, cytoplasmic extracts; Nuc, nuclear extracts.

Figure 3

Immuno-electron microscopy for fukutin in HeLa cells (a, b). Gold particles are seen in the nucleus, predominantly in the heterochromatin, and presumably in the Golgi apparatus and/or endoplasmic reticulum (arrows). N, nucleus.

Figure 4

Results of RT-PCR in HeLa (a) and ZR-75-1 cells (b) after RNAi. In RT-PCR using primers for fukutin, POMGnT1, POMT1 and DG cDNA, amplification products for fukutin are reduced significantly in both cell lines after RNAi, but those for POMGnT1, POMT1 or DG are not. Control, cells only treated with lipofectamine; F697, cells transfected with F697.

Figure 5

Cell morphology (a–c) and results of immunohistochemistry for fukutin (d–i) in HeLa and ZR-75-1 cells after RNAi. Compared with controls (a), HeLa cells exhibit an elongated appearance after transfection of F697 (b). Cells transfected with SNC, siRNA for negative control, do not show any morphological changes (c). Immunohistochemically, fukutin is positive in the nucleus and cytoplasm in control HeLa and ZR-75-1 cells (d, g), and the immunoreaction in the nucleus and cytoplasm is reduced in cells transfected with F697 (e, h). Immunoreaction is also reduced when the primary antibody is pre-absorbed with fukutin protein (f, i). Control, cells only treated with lipofectamine; F697, cells transfected with F697; SNC, cells transfected with SNC.

Figure 6

Results of immunohistochemistry using various antibodies in HeLa and ZR-75-1 cells after RNAi. No clear differences are found in α-DG immunohistochemistry (a–d). Ki-67-positive cells increases after knockdown of fukutin in both HeLa and ZR-75-1 cells (e–h). After knockdown of fukutin, immunoreaction for c-jun becomes stronger in the nucleus and cytoplasm of HeLa cells, (i, j) and increases slightly in ZR-75-1 cells (k, l). There are no apparent differences in c-fos or cyclin D1 (m–t). Control, cells only treated with lipofectamine; F697, cells transfected with F697; D1, cyclin D1.

Figure 7

Results of laminin-binding assay after RNAi. Elongated cells and rounded cells were observed in HeLa cells after inoculation in laminin-coated dishes. Photos show appearance of cells 6 h after inoculation. A ratio of attached cells to non-attached ones was tended to be low in fukutin-suppressed HeLa (a) and ZR-75-1 cells (b). Control, cells only treated with lipofectamine; F697, cells transfected with F697.