Isolation and characterization of a human ileocecal carcinoma cell line (HCT-8) subclone resistant to fluorodeoxyuridine

Zhang Zheng-Gang; Martin Malmberg; M.-B. Yin; Harry K. Slocum; Youcef M. Rustum

doi:10.1016/0006-2952(93)90262-U

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Isolation and characterization of a human ileocecal carcinoma cell line (HCT-8) subclone resistant to fluorodeoxyuridine

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Isolation and characterization of a human ileocecal carcinoma cell line (HCT-8) subclone resistant to fluorodeoxyuridine

Zhang Zheng-Gang, Martin Malmberg, M.-B. Yin, Harry K. Slocum and Youcef M. Rustum

Biochemical pharmacology, Vol.45(5), pp.1157-1164

1993

DOI: 10.1016/0006-2952(93)90262-U

PMID: 8461045

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Abstract

A 5-fluoro-2'-deoxyuridine (FdUrd)-resistant subclone (Fd9XR) of HCT-8 (human ileocecal carcinoma) cells was established by two schedules of drug exposure. Initially, cells were exposed to short-term (3 hr) 100 nM FdUrd repeatedly (9 cycles over 8 months), and cells were then exposed to 10 nM FdUrd continuously. During this latter stage, a colony (Fd9XR) with fast growth rate was isolated, expanded, and characterized with respect to mechanisms of resistance to FdUrd and cross-resistance to other chemotherapeutic agents. Fd9XR cells were 1000-fold resistant to FdUrd, but 3-fold more sensitive to 5-fluorouracil (FUra) than HCT-8 cells. After a 3-hr treatment with FdUrd, Fd9XR cells accumulated 6630-, 69-, and 3.7-fold less fluorodeoxyuridylate (FdUMP), fluorouridine triphosphate (FUTP) and acid-insoluble materials, respectively, than HCT-8 cells. However, when FUra was substituted for FdUrd, Fd9XR cells accumulated 9.2-, 3.1-, and 2.3-fold more FdUMP, FUTP and acid-insoluble materials, respectively, than HCT-8 cells. Fd9XR and HCT-8 were similar in their growth rates, combined pools of 5,10-methylenetetrahydroiolates (5,10-CH 2H 4PteGlu n) and tetrahydrofolates (H 4PTeGlu n), thymidine phosphorylase (TP) activity, and level and activity of thymidylate synthase (TS). In contrast, thymidine kinase (TK) activity of Fd9XR was 0.23 and 0.35% of that of HCT-8, for thymidine (dThd) and FdUrd as substrates, respectively. Furthermore, Fd9XR cells exhibited greater sensitivity to the antifolate TS inhibitor ICI D1694 and to methotrexate (MTX) than HCT-8 cells. In addition, dThd alone and in combination with hypoxanthine did not offer any protection against the cytotoxic effect of ICI D1694 in Fd9XR cells. These results indicate that in Fd9XR cells (1) TK deficiency is the primary mechanism of resistance to FdUrd; (2) the greater sensitivity to FUra was associated with higher pools of FdUMP and FUTP with a subsequently higher level of incorporation into cellular RNA; and (3) antifolate compounds, e.g. ICI D1694 and MTX, could be useful agents in the treatment of FdUrd-resistant tumors associated with decreased TK activity and decreased capacity of utilizing dThd.

FdUrd, 5-fluoro-2'-deoxyuridine

dThd, thymidine

FdUDP, 5-fluoro-2'-deoxyuridine diphosphate

FdUTP, 5-fluoro-2'-deoxyuridine triphosphate

FUra, 5-fluorouracil

TK, thymidine kinase

FdUMP, 5-fluoro-2'-deoxyridylate

TS, thymidylate synthase

MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

Fd9X, a subpopulation of HCT-8 cells surviving 9 cycles of 3-hr exposure to 100 nM FdUrd

FUDP, 5-fluorouridine diphosphate

5,10-CH 2H 4PteGlu n and H 4PteGlU n, 5,10-methylene-tetrahydrofolates and tetrahydrofolates containing n glutamate residues in γ-linkage

DTT, dithiothreitol

dUMP, 2'-deoxyuridylate

PBS, phosphate-buffered saline

FUTP, 5-fluoro-uridine triphosphate

MTX, methotrexate

5-HCOH 4PteGlu, 5-fonnyltetrahydrofolate or leucovorin

TP, thymidine phosphorylase

DMSO, dimethyl sulfoxide

ICI D1694, N-(5-[ N-(3,4-dihydro-2-methyl-4-oxoquinazoline-6-ylmethyl)- N-methylaminol-2-thenoyl)- l-glutamic acid

and Fd9XR, a subclone of Fd9X whose growth rate was among the fastest of all Fd9X cells

FUMP, 5-fluorouridylate

Details

Title: Subtitle: Isolation and characterization of a human ileocecal carcinoma cell line (HCT-8) subclone resistant to fluorodeoxyuridine
Creators: Zhang Zheng-Gang - Roswell Park Cancer Institute
Martin Malmberg - Roswell Park Cancer Institute
M.-B. Yin - Roswell Park Cancer Institute
Harry K. Slocum - Roswell Park Cancer Institute
Youcef M. Rustum - Roswell Park Cancer Institute
Resource Type: Journal article
Publication Details: Biochemical pharmacology, Vol.45(5), pp.1157-1164
Publisher: Elsevier Inc
DOI: 10.1016/0006-2952(93)90262-U
PMID: 8461045
ISSN: 0006-2952
eISSN: 1873-2968
Language: English
Date published: 1993
Academic Unit: Hematology, Oncology, and Blood & Marrow Transplantation; Internal Medicine
Record Identifier: 9984359680702771

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