Comparison of In Vitro Cell Transformation Assay Using Murine Fibroblasts and Human Keratinocytes

Ahn, Junho; Park, Sue Nie; Yum, Yung-Na; Kim, Jiyoung; Lee, Michael

doi:10.5487/tr.2008.24.1.037

Cited by 2 publications

(1 citation statement)

References 22 publications

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“…The in vitro two-stage cell transformation assay (CTA) was performed as described [ 21 ]. Briefly, cells were plated in 6-well plate at 4000 cells/well.…”

Section: Methodsmentioning

confidence: 99%

Paradoxical downregulation of LPAR3 exerts tumor-promoting activity through autophagy induction in Ras-transformed cells

2022

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Background Lysophosphatidic acid receptor 3 (LPAR3) is coupled to Gαi/o and Gα11/q signaling. Previously, we reported that LPAR3 is highly methylated in carcinogen-induced transformed cells. Here, we demonstrate that LPAR3 exhibits malignant transforming activities, despite being downregulated in transformed cells. Methods The LPAR3 knockout (KO) in NIH 3 T3 and Bhas 42 cells was established using the CRISPR/Cas9 system. Both RT-PCR and DNA sequencing were performed to confirm the KO of LPAR3. The cellular effects of LPAR3 KO were further examined by WST-1 assay, immunoblotting analysis, transwell migration assay, colony formation assay, wound scratch assday, in vitro cell transformation assay, and autophagy assay. Results In v-H-ras-transformed cells (Ras-NIH 3 T3) with LPAR3 downregulation, ectopic expression of LPAR3 significantly enhanced the migration. In particular, LPAR3 knockout (KO) in Bhas 42 (v-Ha-ras transfected Balb/c 3 T3) and NIH 3 T3 cells caused a decrease in cell survival, transformed foci, and colony formation. LPAR3 KO led to the robust accumulation of LC3-II and autophagosomes and inhibition of autophagic flux by disrupting autophagosome fusion with lysosome. Conversely, autolysosome maturation proceeded normally in Ras-NIH 3 T3 cells upon LPAR3 downregulation. Basal phosphorylation of MEK and ERK markedly increased in Ras-NIH 3 T3 cells, whereas being significantly lower in LPAR3 KO cells, suggesting that increased MEK signaling is involved in autophagosome–lysosome fusion in Ras-NIH 3 T3 cells. Conclusions Paradoxical downregulation of LPAR3 exerts cooperative tumor-promoting activity with MEK activation through autophagy induction in Ras-transformed cells. Our findings have implications for the development of cancer chemotherapeutic approaches.

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“…The in vitro two-stage cell transformation assay (CTA) was performed as described [ 21 ]. Briefly, cells were plated in 6-well plate at 4000 cells/well.…”

Section: Methodsmentioning

confidence: 99%

Paradoxical downregulation of LPAR3 exerts tumor-promoting activity through autophagy induction in Ras-transformed cells

2022

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Neoplastic Alterations Induced in Mammalian Skin Following Mancozeb Exposure UsingIn VivoandIn VitroModels

Tyagi

George

Bhui

et al. 2011

OMICS: A Journal of Integrative Biology

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Mancozeb, ethylene(bis)dithiocarbamate fungicides, has been well documented in the literature as a multipotent carcinogen, but the underlying mechanism remains unrevealed. Thus, mancozeb has been selected in this study with the objective to decipher the molecular mechanism that culminates in carcinogenesis. We employed two-dimensional gel electrophoresis and mass spectrometry to generate a comparative proteome profile of control and mancozeb (200 mg/kg body weight) exposed mouse skin. Although many differentially expressed proteins were found, among them, two significantly upregulated proteins, namely, S100A6 (Calcyclin) and S100A9 (Calgranulin-B), are known markers of keratinocyte differentiation and proliferation, which suggested their role in mancozeb-induced neoplastic alterations. Therefore, we verified these alterations in the human system by using HaCaT cells as an in vitro model for human skin keratinocyte carcinogenesis. Upregulation of these two proteins upon mancozeb (0.5 μg/mL) exposure in HaCaT cells indicated its neoplastic potential in human skin also. This potential was confirmed by increase in number of colonies in colony formation and anchorage-independent growth assays. Modulation of S100A6/S100A9 targets, elevated phosphorylation of extracellular signal regulated kinase (ERK1/2), Elk1, nuclear factor- kappa B and cell division cycle 25 C phosphatase, and cyclin D1 and cyclooxygenase-2 upregulation was seen. In addition, PD98059 (ERK1/2 inhibitor) reduced cell proliferation induced by mancozeb, confirming the involvement of ERK1/2 signaling. Conclusively, we herein present the first report asserting that the mechanism involving S100A6 and S100A9 regulated ERK1/2 signaling underlies the mancozeb-induced neoplastic potential in human skin.

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Comparison of In Vitro Cell Transformation Assay Using Murine Fibroblasts and Human Keratinocytes

Cited by 2 publications

References 22 publications

Paradoxical downregulation of LPAR3 exerts tumor-promoting activity through autophagy induction in Ras-transformed cells

Paradoxical downregulation of LPAR3 exerts tumor-promoting activity through autophagy induction in Ras-transformed cells

Neoplastic Alterations Induced in Mammalian Skin Following Mancozeb Exposure UsingIn VivoandIn VitroModels

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