Ablation of the ATP-binding cassette transporter, Abca2 modifies response to estrogen-based therapies

Abstract

The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein expressed in oligodendrocytes and Schwann cells, prostate, ovary and macrophages. In cell cultures, ABCA2 over-expression has been linked with resistance to the anticancer agent, estramustine phosphate (EMP; a nor-nitrogen mustard conjugate of estradiol). The present study shows that Abca2 knockout (KO) mice have greater sensitivity to a variety of side effects induced by EMP treatment. Chronic EMP (12×100 mg/kg body weight) produced mortality in 36% of KO mice, but only 7% of age-matched wild type (WT). Side effects of the drug were also more prevalent in the KO mouse. For example, during the first week of EMP treatments, 67% of KO males (compared to 6% of WT males) responded with episodic erectile events. In WT mice, ABCA2 protein localized within pene corpuscles, (which rely on modified Schwann cells for amplification of tactile signals) suggesting that the transporter may function in the erectile process. Endothelial nitric oxide synthase (eNOS; a source of nitric oxide during erectile response) levels were similar in WT and KO male penile tissue. Treatment with 100 mg/kg EMP (once daily for four days) elevated serum estradiol and estrone in both WT and KO. However, the circulating levels of these estrogens were higher in KO mice implying a reduced plasma clearance of estrogens as a consequence of ABCA2 ablation. Consistent with the pro-convulsant effects of estrogens, KO mice also displayed an increased incidence of seizures following EMP (14% vs. 0%). Taken together, these data indicate that ABCA2 deficiency renders mice more sensitive to EMP treatment-induced effects implying that the transporter has a role in regulating EMP transport and/or metabolism.

Fig. 1

Estramustine phosphate treatment protocols.

Fig. 2

Reduced survival in knockout (KO) mice following chronic 6w estramustine phosphate (EMP) treatment. Kaplan-Meyer survival curves of wild type (WT) (solid line) and KO (dashed line) animals treated with 6w schedule of EMP (P = 0.001).

Fig. 3

Lack of hematological toxicities in both wild type (WT) and knockout (KO) mice in response to chronic (6w) estramustine phosphate (EMP) treatment. (A) Total white blood cell counts remained within the normal range throughout the treatment weeks (shaded area indicates normal range for WBC count in mice). No significant changes in body weights were observed in males (B) or females (C). Closed shapes, WT; open shapes, KO. Circles, vehicle treated; triangles, EMP-treated.

Fig. 4

ATP-binding cassette transporter 2 (ABCA2) protein expression and impact of estramustine phosphate (EMP) treatment within male reproductive tissues. Representative localization of ABCA2 in Pacinian corpuscles of (A) wild type (WT); or (B) knockout (KO) mouse pene. (C) ABCA2 expression (see arrow) in helicine arteries of corpus cavernosum of WT and (D) KO mice. KO mouse tissues in (B) and (D) are shown for background staining of ABCA2 polyclonal antibodies. (E) Nitric oxide synthase (NOS) activity is elevated in KO pene compared to WT, but falls short of statistical significance (P = 0.16). NOS activity data are the mean ± standard error of % conversion per μg total protein, shown as percentage of WT-V NOS activity. (F) Prostatic tissue in WT mice shows a significant elevation in apoptotic index following 4d EMP treatment (P < 0.05; defined as the number of TUNEL-positive nuclei per 10 high-powered fields from three to eight mice per group).