Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression

Our plain-English summary of Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression, by H. Kameda, J.I. Risinger, B.B. Han, et al (Molecular and Cellular Biology, 2001).

The study

• Authors: H. Kameda, J.I. Risinger, B.B. Han, S.J. Baek, J.C. Barrett, T. Abe, T. Takeuchi, W.C. Glasgow, T.E. Eling
• Journal: Molecular and Cellular Biology
• Published: 2001;21(20):6895-905
• Cited by: 17 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1(Delta1-103)), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.

In plain English

This was a laboratory study of hamster cells, not people. The authors used two cell clones derived from Syrian hamster embryo cells earlier transformed by asbestos. The article does not name the fiber type. One clone, 10W+8, could still suppress the tumor-forming ability of tumor cells in cell-fusion tests. The other, 10W-1, had lost this ability. The authors studied Gab1, a protein that relays signals from epidermal growth factor (EGF). 10W+8 cells made the normal 100-kDa form of Gab1. Later-passage 10W-1 cells mainly made a shorter 87-kDa form. It lacked its first 103 amino acids, including most of a region called the pleckstrin homology domain, but it still responded to EGF. In soft agar, a test of growth without attachment, later-passage 10W-1 cells formed colonies at 13.6% with EGF and 26.4% with EGF plus insulin. 10W+8 cells reached 3.9% with both. Adding the short form to 10W+8 cells raised EGF-stimulated colony formation to 22.2%, compared with 7.8% for mock-treated cells and 3.1% with normal Gab1 added. The authors concluded that losing this domain may activate Gab1's transforming potential.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-culture experiment using hamster cell clones
• Who was studied: Two asbestos-transformed Syrian hamster embryo (SHE) fibroblast cell clones. 10W+8 (passages 11 to 17) was immortal and could still suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments. 10W-1 had lost that ability and was studied at early passages 11 to 17 (10W-1E) and later passages 25 to 40 (10W-1L). No humans or live animals were studied, apart from an unpublished nude-mouse observation.
• Exposure: The cells had previously been transformed by asbestos (fiber type not stated). In this study, the cells were stimulated with EGF and/or insulin, and the full-length or shortened Gab1 protein was introduced into 10W+8 cells by transfection.
• Main results:
  • Later-passage 10W-1 cells showed an intense 87-kDa form of Gab1. This form was not detected in 10W+8 cells and was slightly detectable in early-passage 10W-1 cells.
  • The 87-kDa form lacked the first 103 amino acids of Gab1, which includes most of the pleckstrin homology (PH) domain.
  • In later-passage 10W-1 cells, the 87-kDa form was tyrosine phosphorylated after EGF stimulation and associated with the EGF receptor.
  • The 87-kDa form also associated with SHP-2 in response to EGF.
  • In soft agar, 10W+8 cells formed no colonies with serum alone and reached a 3.9% colony-forming efficiency with EGF plus insulin.
  • Later-passage 10W-1 cells had a colony-forming efficiency of 13.6% with EGF and 7.7% with insulin.
  • With EGF and insulin combined, later-passage 10W-1 cells reached a colony-forming efficiency of 26.4%.
  • With EGF, mock-transfected 10W+8 cells had a colony-forming efficiency of 7.8%.
  • 10W+8 cells given the shortened Gab1 reached 22.2% with EGF, up to threefold the mock-transfected value. Cells given normal Gab1 had 3.1% (Table 2).
  • Both normal and shortened Gab1 increased DNA synthesis in response to EGF in 10W+8 cells, up to twofold compared with mock-transfected cells.
  • Neither normal nor shortened Gab1 enhanced EGF-dependent MAPK activation in 10W+8 cells.
  • In unpublished work cited by the authors, later-passage 10W-1 cells did not form tumors when injected into nude mice.
• The authors' conclusion: The authors concluded that Gab1 is a key element in EGF signaling and that its transforming potential may be activated by the loss of its amino-terminal PH domain.
• Limitations the authors state:
  • The authors state that why the shortened Gab1, but not full-length Gab1, increased soft-agar colony formation in 10W+8 cells remains to be clarified.
  • Mock-transfected 10W+8 cells formed more colonies than the parent cells. The authors suggest this may be due to the transfection procedure and the many passages during selection.
  • The authors note that Northern and Western analyses appeared less sensitive than RT-PCR for detecting the shortened form.

Related studies

• Silica binds serum proteins resulting in a shift of the dose-response for silica-induced chemokine expression in an alveolar type II cell line — E.G. Barrett, C. Johnston, et al. (1999)
• Structure and interaction of ubiquitin-associated domain of human Fas-associated factor 1 — J. Song, J.K. Park, et al. (2009)
• Mineral fibre toxicity: expression of retinoblastoma (Rb) and phospho-retinoblastoma (pRb) protein in alveolar epithelial and mesothelial cell lines exposed to fluoro-edenite fibres — G. Musumeci, V. Cardile, et al. (2011)
• Post-transcriptional regulation of plasminogen activator inhibitor type-1 expression in human pleural mesothelial cells — S. Shetty, T. Velusamy, et al. (2010)
• LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade — I. Tanaka, H. Osada, et al. (2015)
• Effect of syndecan-1 overexpression on mesenchymal tumour cell proliferation with focus on different functional domains — F. Zong, E. Fthenou, et al. (2010)

Browse return to medical research.