Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins

Abstract

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (-/-) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (-/-) mice]. In spite of the host of functions speculatively attributed to the mdrl-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum-chemical, and immunological parameters were not abnormal in mdr1a/1b (-/-) mice. The high level of mdrlb P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (-/-) mice behaved similarly to the previously analyzed mdr1a (-/-) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (-/-) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (-/-) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.

Figure 1

Targeted disruption of the mdr1b gene. (A) Structure of part of the mdr1b gene, the mdr1b targeting fragment, and the targeted locus. Numbered black boxes represent exons. Transcriptional direction of the neo cassette is indicated. Hatched boxes, 5′ and 3′ probes. Double-headed arrows, position and size of restriction fragments diagnostic for the wild-type (Upper) and correctly targeted (Lower) locus. Restriction sites: Nt, NotI; S, SmaI; K, KpnI; A, ApaI; N, NcoI; B, BamHI. (Scale bar = 2 kb.) (B) Secondary targeting of the mdr1b locus (neo cassette) in a chromosome containing an mdr1a disruption (hygro cassette). Double-headed arrows indicate position and size of the MluI restriction fragments diagnostic for linked (≈550 kb) and unlinked (≈1000 kb) disruption of the mdr1a and mdr1b genes as detected with the hygro probe (left hatched box). Open boxes, genes and selectable cassettes. The 3′ end of the mdr1a gene is not yet mapped. (MluI) indicates a partially cut MluI site (likely due to partial CG methylation). Other designations are as in A.

Figure 2

Absence of P-gp in adrenal glands of mdr1a/1b (−/−) mice. Protein immunoblot analysis of adrenal gland membrane fractions (10 μg per lane) using monoclonal antibody C219. mdr1a/1b genotypes and protein marker sizes are indicated. f, female; m, male. Lane 1b contains a membrane fraction isolated from mdr1b-transfected SW1573 (lung cancer) cells.

Figure 3

Rhodamine efflux from wild-type, mdr1a (−/−), mdr1b (−/−), and mdr1a/1b (−/−) bone marrow cells. Partially purified (WGA⁺/15–1.1⁻) bone marrow progenitor cells were loaded with rhodamine 123. Subsequent efflux in rhodamine-free medium was measured by fluorescence-activated cell sorting. Qualitatively comparable results were obtained in three out of three independent experiments. (○), wild-type cells; (•), mdr1a (−/−) cells; (Δ), mdr1b (−/−) cells; (▿), mdr1a/1b (−/−) cells; (▴), wild-type granulocytes.