Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart

A. Rosati, G. Decorti, F. B. Klugmann, L. Candussio, M. Granzotto, M. Melato, T. Giraldi

Research output: Contribution to journalArticle

Abstract

P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy. These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin. Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP. The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression. P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used. A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines. The uptake was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp. Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.

Original languageEnglish
Pages (from-to)3403-3410
Number of pages8
JournalAnticancer Research
Volume20
Issue number5 B
Publication statusPublished - 2000

Fingerprint

P-Glycoproteins
P-Glycoprotein
Kidney
Cell Line
LLC-PK1 Cells
Doxorubicin
Rhodamine 123
Verapamil
Proximal Kidney Tubule
Propidium
Multiple Drug Resistance
Tumor Cell Line
Antineoplastic Agents
Fluorescent Antibody Technique
Swine
Fluorescence
Staining and Labeling
Liver
Therapeutics
Neoplasms

Keywords

  • Cells
  • Cytofluorimetry
  • Doxorubicin
  • Immunofluorescence
  • LLC-PK
  • LLC-PK/ADR cells
  • Multidrug resistance
  • Multidrug resistance related protein
  • P-glycoprotein
  • Rhodamine 123

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Rosati, A., Decorti, G., Klugmann, F. B., Candussio, L., Granzotto, M., Melato, M., & Giraldi, T. (2000). Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart. Anticancer Research, 20(5 B), 3403-3410.

Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart. / Rosati, A.; Decorti, G.; Klugmann, F. B.; Candussio, L.; Granzotto, M.; Melato, M.; Giraldi, T.

In: Anticancer Research, Vol. 20, No. 5 B, 2000, p. 3403-3410.

Research output: Contribution to journalArticle

Rosati, A, Decorti, G, Klugmann, FB, Candussio, L, Granzotto, M, Melato, M & Giraldi, T 2000, 'Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart', Anticancer Research, vol. 20, no. 5 B, pp. 3403-3410.
Rosati A, Decorti G, Klugmann FB, Candussio L, Granzotto M, Melato M et al. Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart. Anticancer Research. 2000;20(5 B):3403-3410.
Rosati, A. ; Decorti, G. ; Klugmann, F. B. ; Candussio, L. ; Granzotto, M. ; Melato, M. ; Giraldi, T. / Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart. In: Anticancer Research. 2000 ; Vol. 20, No. 5 B. pp. 3403-3410.
@article{9dd1c949d5da42e198c9f3845b6197fc,
title = "Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart",
abstract = "P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy. These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin. Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP. The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression. P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used. A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines. The uptake was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp. Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.",
keywords = "Cells, Cytofluorimetry, Doxorubicin, Immunofluorescence, LLC-PK, LLC-PK/ADR cells, Multidrug resistance, Multidrug resistance related protein, P-glycoprotein, Rhodamine 123",
author = "A. Rosati and G. Decorti and Klugmann, {F. B.} and L. Candussio and M. Granzotto and M. Melato and T. Giraldi",
year = "2000",
language = "English",
volume = "20",
pages = "3403--3410",
journal = "Anticancer Research",
issn = "0250-7005",
publisher = "International Institute of Anticancer Research",
number = "5 B",

}

TY - JOUR

T1 - Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart

AU - Rosati, A.

AU - Decorti, G.

AU - Klugmann, F. B.

AU - Candussio, L.

AU - Granzotto, M.

AU - Melato, M.

AU - Giraldi, T.

PY - 2000

Y1 - 2000

N2 - P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy. These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin. Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP. The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression. P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used. A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines. The uptake was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp. Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.

AB - P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy. These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin. Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP. The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression. P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used. A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines. The uptake was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp. Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.

KW - Cells

KW - Cytofluorimetry

KW - Doxorubicin

KW - Immunofluorescence

KW - LLC-PK

KW - LLC-PK/ADR cells

KW - Multidrug resistance

KW - Multidrug resistance related protein

KW - P-glycoprotein

KW - Rhodamine 123

UR - http://www.scopus.com/inward/record.url?scp=0033635037&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033635037&partnerID=8YFLogxK

M3 - Article

C2 - 11131640

AN - SCOPUS:0033635037

VL - 20

SP - 3403

EP - 3410

JO - Anticancer Research

JF - Anticancer Research

SN - 0250-7005

IS - 5 B

ER -