Identification of a novel insulin-like growth factor binding protein gene homologue with tumor suppressor like properties

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Abstract

Here we report the identification of a new insulin-like growth factor binding protein homologue, provisionally designated insulin-like growth factor binding related protein-4 (IGFBP-rP4). IGFBP-rP4 was found to be most closely related to IGFBP-7 with 52% amino acid homology and 43% amino acid identity, and shares a similar domain structure. Semi-quantitative RT-PCR expression analysis demonstrated a pattern of downregulation of this gene in multiple tumor samples including lung and colon cancer, compared to matched adjacent normal tissue. Western blotting revealed a protein of approximately 38 kDa expressed in both the cell pellet and secreted into the supernatant of transiently transfected Cos-7 cells. Cos-7 supernatants containing IGFBP-RP4 protein were observed to suppress the growth of HeLa cells in culture compared to vector controls. IGFBP-RP4 directly transiently transfected into HeLa cells also further confirmed the growth suppressive properties of this protein. Together these data suggest that IGFBP-RP4 may be a novel putative tumor suppressor protein.

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Materials and methods

Cell lines and tissues. All cell lines used in this study were obtained from the American Type Culture Collection (ATCC) and included the Cos-7 (an African Green Monkey kidney cell line) and HeLa cells (human cervical carcinoma cells). Culturing of cell lines was performed using standard protocols. All healthy tissue total RNAs were obtained from either Ambion or Clontech. Cancer patient tissues were obtained from the Cooperative Human Tissue Network (CHTN) or from Clontech and Ambion where

IGFBP-RP4 gene characterization

The IGFBP-RP4 cDNA and protein sequences are shown in Fig. 1. IGFBP-RP4 is a protein of 278 amino acids with a signal peptide and conserved motifs found in the IGFBP family of proteins. The signal peptide runs from the N-terminus through G27, which is where the signal peptide cleavage site is predicted. Additionally, from amino acid D61 through S76, there is a conserved IGFBP family signature as predicted by the eMatrix software program. The Pfam software program predicts a Kazal

Discussion

A number of IGFBP family members have been suggested to have tumor suppressor like properties. IGFBP-3 has been shown to suppress the growth of breast cancer cells and the prostate cancer cell line PC-3 [1]. IGFBP-7, the family member that shares the greatest sequence similarity to IGFBP-rP4, had been demonstrated to suppress the growth of HeLa, P19 (murine embryonic carcinoma cells), and Saos-2 (osteosarcoma cells) when recombinant IGFBP-7 protein was added to the culture medium [11].

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