v-Ras and v-Raf Block Differentiation of Transformable C3H10T1/2-Derived Preadipocytes at Lower Levels Than Required for Neoplastic Transformation

Raptis, Leda; Brownell, Heather L.; Lu, Ying; Preston, Thomas J.; Narsimhan, Radha P.; Anderson, Stephen; Schaefer, Erik; Haliotis, Tina

doi:10.1006/excr.1997.3646

Cited by 13 publications

(21 citation statements)

References 34 publications

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“…lines 25B8 and 24D4) express ϳ30% the c-Ras levels present in the parental 10T 1 ⁄2 and they differentiate into adipocytes very effectively (22). A number of lines were produced which permanently express the ras-antisense and Hsp90N, using the same plasmid and hygromycin resistance selection as described before (21,22).…”

Section: Methodsmentioning

confidence: 99%

“…Induction and Quantitation of Adipocytic Differentiation-Standard procedures described before were followed (22,26). Briefly, ϳ10 3 cells were seeded into each well of a 24-well Linbro plate (Corning) in 10% fetal calf serum.…”

Section: Methodsmentioning

confidence: 99%

“…This medium was changed 48 h later to medium containing 10 g/ml insulin and 10% fetal calf serum. Lipid droplets appeared ϳ10 days later and stained with Oil Red-O as described (22,26). To increase the attachment of transformed cells to the culture plates, cells were plated onto Cell-Tak, a polyphenolic protein extracted from a marine mussel, according to the manufacturer's instructions (Collaborative Research).…”

Section: Methodsmentioning

confidence: 99%

“…To this effect, we used a panel of mouse 10T 1 ⁄2-derived lines where c-Ras levels had been reduced to ϳ30% of the normal through the introduction of an antisense message (e.g. line 25B8 (22,25)). These cells were transfected with the same construct containing the Hsp90N sequence as for the F111 cells above.…”

Section: Hsp90n Transfection In Rat F111 Fibroblasts Leads To Raf Actmentioning

confidence: 99%

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The Role of Hsp90N, a New Member of the Hsp90 Family, in Signal Transduction and Neoplastic Transformation

Grammatikakis¹,

Vultur²,

Ramana³

et al. 2002

Journal of Biological Chemistry

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The 90-kDa heat shock protein (Hsp90), the target of the ansamycin class of anti-cancer drugs, is required for the conformational activation of a specific group of signal transducers, including Raf-1. In this report we have identified a 75-kDa Raf-associated protein as Hsp90N, a novel member of the Hsp90 family. Intriguingly, the ansamycin-binding domain is replaced in Hsp90N by a much shorter, hydrophobic sequence, preceded by a putative myristylation signal. We demonstrate that, although much less abundant, Hsp90N binds Raf with a higher affinity than Hsp90. In sharp contrast to Hsp90, Hsp90N does not associate with p50 cdc37 , the Hsp90 kinase cofactor. Hsp90N was found to activate Raf in transiently transfected cells, while Rat F111 fibroblasts stably transfected with Hsp90N exhibited elevated activity of the Raf and downstream ERK kinases. This may be due to Raf binding to myristylated Hsp90N, followed by Raf translocation to the membrane. To examine whether Hsp90N could therefore substitute for Ras in Raf recruitment to the cell membrane, Hsp90N was transfected in c-Ras-deficient, 10T 1 ⁄2-derived preadipocytes. Our results indicate that, as shown before for activated Ras or Raf, the introduction of even low levels of Hsp90N through transfection in c-Ras-deficient preadipocytes causes a dramatic block of differentiation. Higher levels of Hsp90N expression resulted in neoplastic transformation, including interruption of gap junctional, intercellular communication, and anchorage-independent proliferation. These results indicate that the observed activation of Raf by Hsp90N has a profound biological effect, which is largely c-Ras-independent. With the recent finding that p50 cdc37 is tumorigenic in transgenic mice, these results reinforce the intriguing observation that the family of heat shock proteins represents a novel class of molecules with oncogenic potential.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Hsp90n Transfection In Rat F111 Fibroblasts Leads To Raf Actmentioning

confidence: 99%

See 2 more Smart Citations

The Role of Hsp90N, a New Member of the Hsp90 Family, in Signal Transduction and Neoplastic Transformation

Grammatikakis¹,

Vultur²,

Ramana³

et al. 2002

Journal of Biological Chemistry

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“…In a series of studies, it was shown that expression of oncogenic Ras in 3T3-L1 preadipocytes induces adipocyte differentiation, whereas expression of dominant-negative Ras abrogates adipose conversion of these cells (4,5). In contrast, it was reported that expression of an activated Ras mutant abolished differentiation of both C3H10T1/2 and certain subclones of 3T3-L1 cells (6,7). Terminal adipocyte differentiation is promoted by or dependent on the activity of a number of kinases, most notably kinases of the phosphatidylinositol 3-kinase-protein kinase B (PKB/Akt) pathway (8,9) and kinases leading to activation of p38 mitogen-activated protein kinases (MAPKs) (10,11).…”

mentioning

confidence: 93%

Deregulated MAPK Activity Prevents Adipocyte Differentiation of Fibroblasts Lacking the Retinoblastoma Protein

Hansen

Petersen

Jørgensen

et al. 2002

Journal of Biological Chemistry

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A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity. Ras-dependent signaling plays a significant, although incompletely understood, role in adipocyte differentiation, because activated Ras has been reported to either promote or inhibit adipogenesis depending on the cellular context. In various cell types activation of Ras leads to activation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein kinase B (PKB)/Akt, which exert opposing effects on adipogenesis, with ERK1/2 inhibiting and PKB/Akt promoting terminal differentiation. Here we report that the levels of activated ERK1/2 and PKB/Akt are significantly increased in pRB-deficient MEFs both before and after the addition of adipogenic inducers. Consistently, we detected higher levels of activated Ras in MEFs lacking pRB. Suppression of ERK1/2 activation by the MEK inhibitor UO126 restored the ability of pRB-deficient MEFs to undergo adipocyte differentiation, as manifested by expression of adipocyte marker genes and lipid accumulation. Furthermore and reflecting the elevated levels of activated PKB/Akt in the pRB-deficient MEFs, differentiation proceeded in an insulin-independent manner. In conclusion, we suggest that pRB plays a pivotal role in adipogenesis by suppressing MAPK activity.The conversion of fibroblast-like precursor cells to fully differentiated adipocytes is a process controlled by a complex network of signaling pathways and is tightly regulated by numerous transcription factors, including members of the peroxisome proliferator-activated receptor (PPAR) 1 and CCAAT/ enhancer-binding protein (C/EBP) families, and the adipocyte determination and differentiation-dependent factor-1/sterol regulatory element-binding protein-1 (for review, see Refs.

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Mitogen‐activated protein kinase expression and activation does not differentiate benign from malignant mesothelial cells

Vintman

Nielsen

Berner

et al. 2005

Cancer

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BACKGROUND In vitro studies of malignant mesothelioma (MM) cells have suggested activation of mitogen‐activated protein kinase (MAPK) in response to asbestos exposure. The objective of this study was to investigate protein expression (level) and phosphorylation status (activity) of the extracellular‐regulated kinase (ERK), the c‐Jun amino‐terminal kinase (JNK), and the high‐osmolarity glycerol response kinase (p38) in vivo through the analysis of fresh frozen reactive mesothelium (RM) and MM specimens. METHODS MAPK levels were analyzed in 36 fresh‐frozen MM specimens (32 effusions, 4 biopsies) and in 14 RM specimens (all effusions) using immunoblotting with antibodies detecting the total (pan‐) and activated (phospho‐) fraction (p‐) of ERK, JNK, and p38. Values for pan‐MAPK and p‐MAPK expression and the p‐MAPK/pan‐MAPK ratio in MM and RM specimens were compared. Results were corroborated using immunocytochemistry for p‐ERK, p‐JNK, and p‐38 in selected specimens. RESULTS Pan‐ERK, pan‐JNK, and pan‐p38 expression was found frequently in both MM specimens (35 of 36 specimens) and RM specimens (14 of 14 specimens) using immunoblotting, with comparable findings for activated p‐p38 (34 of 36 MM specimens, 13 of 14 RM specimens). Activation of p‐ERK (27 of 36 MM specimens, 10 of 14 RM specimens) and p‐JNK (25 of 36 MM specimens, 10 of 14 RM specimens) was less frequent. Pan‐ERK (P = 0.016), pan‐JNK (P = 0.004), pan‐p38 (P = 0.012), and p‐ERK (P = 0.02) expression levels were higher in MM specimens from female patients. Pan‐p38 expression levels also were higher in peritoneal MM specimens (P = 0.019). MM and RM showed similar MAPK expression, activation, and activation ratios (Mann–Whitney test; P > 0.05). Immunocytochemistry localized MAPK to MM and RM cells. CONCLUSIONS The current results provided the first evidence of in vivo activation of MAPK in clinical MM and RM. The similar values in these two cell types suggest that MAPK may not be involved in the transformation of benign to malignant mesothelium, thus bringing into question the validity of using MAPKs as molecular therapeutic targets in patients with MM. Cancer 2005. © 2005 American Cancer Society.

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v-Ras and v-Raf Block Differentiation of Transformable C3H10T1/2-Derived Preadipocytes at Lower Levels Than Required for Neoplastic Transformation

Cited by 13 publications

References 34 publications

The Role of Hsp90N, a New Member of the Hsp90 Family, in Signal Transduction and Neoplastic Transformation

The Role of Hsp90N, a New Member of the Hsp90 Family, in Signal Transduction and Neoplastic Transformation

Deregulated MAPK Activity Prevents Adipocyte Differentiation of Fibroblasts Lacking the Retinoblastoma Protein

Mitogen‐activated protein kinase expression and activation does not differentiate benign from malignant mesothelial cells

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