The ability of hypoxia to modify the gene expression of thymidylate synthase in tumour cells in vivo.
OBJECTIVE
Hypoxic cells in tumours are resistant to 5-fluorouracil (5-FU). This in vivo study investigated the ability of hypoxia to regulate the gene expression of thymidylate synthase (TS), the target enzyme of 5-FU.


METHODS
C3H mammary carcinomas, grown in the feet of female CDF1 mice, were used for all experiments. Mice were placed in a 10% oxygen environment for various time periods and the tumour oxygen status was determined with an Eppendorf oxygen electrode. The animals were then injected with BrdU (100 mg/kg, i.p.). Tumours were excised and immediately frozen (-80 degrees C) until isolation of total RNA. The mRNA was reversibly transcribed to complementary DNA and the resulting cDNA amplified in a multiplex PCR reaction, with beta-actin as the internal reference gene.


RESULTS
One hour of low oxygen breathing made tumours significantly more hypoxic. This increase was maintained for a maximum incubation period of 48 h. In the same tumours, no change in TS gene expression was seen with up to 3 h of low oxygen breathing. At longer times it decreased, reaching significance at 12-24 h and remaining at this lower level for up to 48 h. BrdU labelling was significantly reduced after breathing low O2 for 24 h (p = 0.001).


CONCLUSIONS
Hypoxia-induced down-regulation of TS gene expression was observed. This would be expected to make hypoxic tumour cells more sensitive to 5-FU. Other mechanisms must be responsible for the previously reported resistance to this drug.
Ehrnrooth
E
E
Department of Oncology, Aarhus University Hospital, Denmark. evae@dadlnet.dk
von der Maase
H
H
Sørensen
B S
BS
Poulsen
J H
JH
Horsman
M R
MR
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004351:Drug Resistance; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology; D015870:Gene Expression; D000860:Hypoxia / Q000201:enzymology* / Q000235:genetics*; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology* / Q000235:genetics* / Q000628:therapy; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics*
D000964:Antimetabolites, Antineoplastic; D017931:DNA Primers; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D013940:Thymidylate Synthase; D005472:Fluorouracil