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Multigenerational study of chemically induced cytotoxicity and proliferation in cultures of human proximal tubular cells.

Lash LH, Putt DA, Benipal B - Int J Mol Sci (2014)

Bottom Line: Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells.P1 and P2 cells were most sensitive to DCVC-induced apoptosis.DCVC also increased cell proliferation most prominently in P1 and P2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA. l.h.lash@wayne.edu.

ABSTRACT
Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1-P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model.

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Effects of DCVC and passage number on p53 expression in hPT cells. Cells (P0–P4) were grown on collagen-coated, T-25 flasks to approximately 80% confluence. Cells were incubated for 24 h with either media (Con = Control) or 100 µM DCVC. Protein expression was quantified by Western blot analysis with a mouse monoclonal antibody raised against the N-terminus of human p53 (Mr 53 kDa), using alkaline phosphatase staining (A) and laser scanning densitometry (B). *, Significantly different (p < 0.05) from the corresponding control.
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ijms-15-21348-f007: Effects of DCVC and passage number on p53 expression in hPT cells. Cells (P0–P4) were grown on collagen-coated, T-25 flasks to approximately 80% confluence. Cells were incubated for 24 h with either media (Con = Control) or 100 µM DCVC. Protein expression was quantified by Western blot analysis with a mouse monoclonal antibody raised against the N-terminus of human p53 (Mr 53 kDa), using alkaline phosphatase staining (A) and laser scanning densitometry (B). *, Significantly different (p < 0.05) from the corresponding control.

Mentions: p53 is another cytoprotective protein that regulates cell growth, repair, and apoptosis in mammalian kidney in response to a variety of stresses [22,23,24]. p53 protein levels were readily detected and were generally maintained from P0 through P4 (Figure 7). Although previous studies in P0 hPT cells showed marked induction of p53 by DCVC [12], DCVC in the present study slightly decreased p53 levels in P0, P1, and P4 cells but had little effect in other generations.


Multigenerational study of chemically induced cytotoxicity and proliferation in cultures of human proximal tubular cells.

Lash LH, Putt DA, Benipal B - Int J Mol Sci (2014)

Effects of DCVC and passage number on p53 expression in hPT cells. Cells (P0–P4) were grown on collagen-coated, T-25 flasks to approximately 80% confluence. Cells were incubated for 24 h with either media (Con = Control) or 100 µM DCVC. Protein expression was quantified by Western blot analysis with a mouse monoclonal antibody raised against the N-terminus of human p53 (Mr 53 kDa), using alkaline phosphatase staining (A) and laser scanning densitometry (B). *, Significantly different (p < 0.05) from the corresponding control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264229&req=5

ijms-15-21348-f007: Effects of DCVC and passage number on p53 expression in hPT cells. Cells (P0–P4) were grown on collagen-coated, T-25 flasks to approximately 80% confluence. Cells were incubated for 24 h with either media (Con = Control) or 100 µM DCVC. Protein expression was quantified by Western blot analysis with a mouse monoclonal antibody raised against the N-terminus of human p53 (Mr 53 kDa), using alkaline phosphatase staining (A) and laser scanning densitometry (B). *, Significantly different (p < 0.05) from the corresponding control.
Mentions: p53 is another cytoprotective protein that regulates cell growth, repair, and apoptosis in mammalian kidney in response to a variety of stresses [22,23,24]. p53 protein levels were readily detected and were generally maintained from P0 through P4 (Figure 7). Although previous studies in P0 hPT cells showed marked induction of p53 by DCVC [12], DCVC in the present study slightly decreased p53 levels in P0, P1, and P4 cells but had little effect in other generations.

Bottom Line: Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells.P1 and P2 cells were most sensitive to DCVC-induced apoptosis.DCVC also increased cell proliferation most prominently in P1 and P2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA. l.h.lash@wayne.edu.

ABSTRACT
Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1-P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model.

Show MeSH
Related in: MedlinePlus