Phenotypic evolution of cells resistant to bromodeoxyuridine

Area of Science:

• Cell biology
• Genetics
• Biochemistry

Background:

• Bromodeoxyuridine (BrdUrd) is a thymidine analog used to study DNA synthesis and cell proliferation.
• Thymidine kinase (TK) is a crucial enzyme in DNA precursor metabolism, phosphorylating thymidine and BrdUrd.
• Drug resistance mechanisms in mammalian cells are complex and can involve multiple genetic and epigenetic alterations.

Purpose of the Study:

• To investigate the mechanisms of bromodeoxyuridine (BrdUrd) resistance and thymidine kinase (TK) deficiency development in V79 Chinese hamster cells.
• To characterize the phenotypic changes associated with prolonged BrdUrd exposure.
• To explore the role of BrdUrd as both a selective and inductive agent in cellular evolution.

Main Methods:

• Induction of drug resistance using spontaneous and drug-induced mutations in V79 cells.
• Isolation of partially resistant clones via one-step selection in BrdUrd.
• Long-term culture of tolerant populations in BrdUrd-containing medium.
• Measurement of thymidine kinase activity and relative plating efficiency in hypoxanthine/aminopterin/thymidine (HAT) medium.
• Clonal analysis to assess phenotypic modifications.

Main Results:

• Prolonged BrdUrd exposure induced a gradual phenotypic transition, leading to complete BrdUrd resistance and thymidine kinase deficiency.
• Thymidine kinase activity declined logarithmically over 8-10 weeks, correlating with increased growth rate.
• Colony formation in HAT medium decreased exponentially for at least 18 weeks post-TK deficiency.
• Phenotypic modifications were continuous or multistep, occurring in most cells maintained in BrdUrd.

Conclusions:

• Bromodeoxyuridine (BrdUrd) can act as both an inductive and selective agent, driving the evolution of drug-resistant cell populations.
• The development of BrdUrd resistance and thymidine kinase deficiency is a complex, multistep process.
• New models are required to explain the progressive evolution of these cellular phenotypes.