Abstract

The aim of this project is to determine the mechanism for entry of the anti-cancer drug cis-diamminedichloroplatinum II (cisplatin) into cancer cells, and how this entry is affected in drug-resistant cells. Drug-sensitive and drug-resistant liver carcinoma cells were incubated for different times (0.5 hr, 1 hr, 2 hr, 4 hr) with 400 micromolar cisplatin and 100 micrograms per ml ferric chloride. Cells were pelleted, rapidly frozen in liquid ethane and cryosectioned. Energy- dispersive x-ray microanalysis showed that ion gradients and elemental distributions were disrupted due to mechanical damage in cells that had been scraped off culture dishes. It was possible to avoid this problem by using the enzyme trypsin to release the cells more gently from the substrate. In the absence of cisplatin, cells prepared in this way were found to have normal Na/K gradients across the plasma membrane. We now plan to continue our experiments on treated cells using x-ray microanalysis to co-localize cisplatin and iron. - cisplatin, x-ray microanalysis, rapid-freezing

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Intramural Research (Z01)
Project #
1Z01OD010491-02
Application #
6290698
Study Section
Special Emphasis Panel (BE)
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Office of the Director, National Institutes of Health
Department
Type
DUNS #
City
State
Country
United States
Zip Code

  Publications

Licht, T; Goldenberg, S K; Vieira, W D et al. (2000) Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice. Gene Ther 7:348-58
Lee, C G; Ramachandra, M; Jeang, K T et al. (2000) Effect of ABC transporters on HIV-1 infection: inhibition of virus production by the MDR1 transporter. FASEB J 14:516-22
Shen, D W; Goldenberg, S; Pastan, I et al. (2000) Decreased accumulation of [14C]carboplatin in human cisplatin-resistant cells results from reduced energy-dependent uptake. J Cell Physiol 183:108-16
Hafkemeyer, P; Licht, T; Pastan, I et al. (2000) Chemoprotection of hematopoietic cells by a mutant P-glycoprotein resistant to a potent chemosensitizer of multidrug-resistant cancers. Hum Gene Ther 11:555-65
Booth, C L; Pulaski, L; Gottesman, M M et al. (2000) Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein. Biochemistry 39:5518-26
Hafkemeyer, P; Brinkmann, U; Gottesman, M M et al. (1999) Apoptosis induced by Pseudomonas exotoxin: a sensitive and rapid marker for gene delivery in vivo. Hum Gene Ther 10:923-34
Licht, T; Aran, J M; Goldenberg, S K et al. (1999) Retroviral transfer of human MDR1 gene to hematopoietic cells: effects of drug selection and of transcript splicing on expression of encoded P-glycoprotein. Hum Gene Ther 10:2173-85
Moscow, J A; Huang, H; Carter, C et al. (1999) Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy. Blood 94:52-61
Hrycyna, C A; Ramachandra, M; Germann, U A et al. (1999) Both ATP sites of human P-glycoprotein are essential but not symmetric. Biochemistry 38:13887-99
Aran, J M; Pastan, I; Gottesman, M M (1999) Therapeutic strategies involving the multidrug resistance phenotype: the MDR1 gene as target, chemoprotectant, and selectable marker in gene therapy. Adv Pharmacol 46:1-42

Showing the most recent 10 out of 16 publications