Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells

Abstract

Full length mRNA to a drug-inhibited cell surface NADH oxidase, tNOX or ENOX2, is present in both non-cancer and cancer cells but is translated only in cancer cells as alternatively spliced variants. ENOX2 is a growth-related protein of the external plasma membrane surface that is shed into the circulation and is inhibited by a series of quinone site inhibitors with anticancer activity. To test the possibility that ENOX2 expression might be important to early stages of non-cancer cell development, the expression of the protein was monitored in chicken embryos during their development. Polyclonal antisera to a 34 kDa human serum form of ENOX2 cross-immunoreactive with the drug-responsive NADH oxidase of chicken hepatoma cells was used. The protein was identified based on drug-responsive enzymatic activities and analyses by western blots. The drug-responsive activity was associated with plasma membranes and sera of early chicken embryos and with chicken hepatoma plasma membranes but was absent from plasma membranes prepared from livers or from sera of normal adult chickens and from late embryo stages. The findings suggest that ENOX2 may fulfill some functions essential to the growth of early embryos which are lost in late embryo stages and absent from normal adult cells but which then reappear in cancer.

Fig. 1

Western blot analysis following 12% SDS-PAGE of immunoprecipitates of isolated plasma membranes from chicken hepatoma cells (H) and chicken livers (L) with immune (a) or preimmune (b) sera. Antisera (rabbit) were used with a dilution of 1:1,000 in PBS. Alkaline phosphatase labeled anti-rabbit antibody was used as a secondary antibody with a dilution of 1:5,000 in TBS-T. a Arrows indicate a ca. 34 kDa component in plasma membrane of hepatoma cells (H) reactive with antisera and not observed with chicken livers (L) either with antisera (a) or with preimmune sera (b)

Fig. 2

Western blot analysis of stages 3–40 of chicken embryo homogenates with polyclonal antibody to 34 kDa ENOX2 isolated from sera of cancer patients as described by Chueh et al. [19] following separation on 12% SDS-PAGE and transferred to nitrocellulose (montage of 3 blots as indicated). The blots were incubated with antisera (rabbit) diluted 1:1,000. Alkaline phosphatase-conjugated antirabbit second antibody was at a dilution of 1:25,000 and incubated with NBT-BCIP. Immunoreactive 34 kDa (upper) and 32 kDa (lower) components are indicated by the arrows. The immunoreactive band at 34 ± 1 kDa was most prevalent. The immunoreactive band at 32 kDa and a faint band at 29 kDa in stage 38 embryos represent processing intermediates (see text)

Fig. 3

NADH oxidase activity measured with plasma membrane preparations from different stages of chicken embryos in the absence (filled circle) or presence of 1 μM (open circle) or 100 μM (open triangle) capsaicin. Only the stages represented here provided sufficient embryo material to allow for isolation of plasma membranes. Values are averages of three determinations ± standard deviations