Understanding the roles of the Reprimo family of proteins in regulating cell behaviour in normal and cancer cells

Abstract

The human Reprimo gene family comprises two genes, RPRM and RPRML. Lack of expression of the RPRM gene has been detected in many human cancer cell lines and tissues.

In this study, an immunochemical analysis of the human RPRM was performed. This showed that RPRM could be detected as a FLAG-tagged protein in transfected 293T cells by Western blotting using either an anti-FLAG antibody or (at greatly reduced efficiency) a polyclonal antibody against RPRM. Subcellular fractionation experiments indicated that transiently-expressed RPRM is mainly present in the cytoplasmic fraction of transfected cells, however fractionation of the cytoplasm into cytosol and membranes revealed a predominant association of the 18kDa RPRM form with intracellular membranes. Analysis of several human cell lines for the endogenous RPRM forms also revealed a membrane location of the 18kDa RPRM form. Localisation of RPRM by immunocytochemistry also revealed it to be associated with membranous structures, that co-localised with the Golgi/endoplasmic reticulum. Immunohistochemical studies on normal and cancerous human tissues (including breast, pancreas and colon) provided further evidence for a membrane location of RPRM.

Studies were performed on the reduction of the endogenous RPRM forms by RNA interference and the impact of RPRM reduction on the cell cycle. These studies revealed small, although significant effects of RPRM on the G1 to S and the of G2 to M phase transition. Over-expression of RPRM by transient transfection of MCF7 breast cancer cells or 293T kidney cells resulted in a block to the G2 to M phase.

In summary, RPRM exists in a number of forms related by differential posttranslational modification, probably principally N-glycosylation. It has a major location with intracellular membranes and influences the cell cycle in different ways, dependent on the level of RPRM expression. However the link between RPRM membrane association and the cell cycle remains to be elucidated.