Efforts to develop anticancer therapies based on suicide transgenes generally focus on prodrug activating enzymes [
38] combined with effective targeting of pathological cells. We have been studying αGalT gene expression and the very high efficiency of natural anti-αGal antibodies in inducing complement-mediated cell killing [
39,
40] in the field of hyperacute xenograft rejection. We attempted to take advantage of this constitutive immune system to target tumor cells. Several authors have demonstrated the efficacy of natural anti-αGal antibodies for destruction of tumor cells [
7‐
10]. Indeed, the high numbers of circulating oligosaccharides bearing αGal epitopes are responsible for constant booster immunizations. This may explain the high plasma level (1% of IgG) of anti-αGal antibodies in humans [
4] and their constant
de novo synthesis in αGalT knockout mice [
41,
42]. Natural anti- αGal antibodies are highly cytotoxic and cytolytic as the result of highly efficient complement activation, and this results in hyperacute rejection of xenografts [
2,
40]. They have also been shown capable of protecting αGalT-deficient mice against engrafted αGal
+ colon cancer cells [
9].
The purpose of this study was the targeting of tumor cells known to overexpress c-erbB-2 using a selected form of its promoter p
Neu to drive an active αGalT. This approach was designed to take advantage of the effective antibodies preexisting in all humans. In a similar study, a derived form of the human telomerase promoter was shown to render human pancreatic carcinoma cells susceptible to αGal/complement-mediated cell killing [
43]. We selected p
Neu because it has been well characterized and is overexpressed in a variety of tumors [
16,
44]. Because definition of a precise p
Neu sequence with well-restricted activation in tumor cells remains uncertain, however, we analyzed several forms of this promoter. The shortest form, p
Neu209, which comprises the only PEA3 motif adjacent to the TATA box plus two SP1 sites and one AP-2 site downstream of the transcription start site, promoted very weak αGal expression. The minimal forms, p
Neu392 and p
Neu250, were equally capable of selectively inducing αGalT in breast tumor cells compared with HEK-293 cells. We thus concluded that the Ap-2 and SP1 motifs downstream of the transcription start site (Fig.
2) were not essential. The noticeable absence of a CCAAT box in p
Neu209 probably explains its disrupted activity because in cells over-expressing c-erbB-2, the CCAAT box is up-regulated rather than the TATAA box [
45]. Further studies thus focused on comparing p
Neu250 with the longest form, p
Neu664. This last form contains several PEA3, NF-
kB, HER2 transcription factor (HTF) and SP1 sites upstream of the minimal p
Neu250. The role played by the Ets family and activator protein-2 (AP-2) factors has been extensively studied in breast tumor cells. While the AP-2 binding site was present in both p
Neu250 and p
Neu664, the main difference between these forms was the presence of three additional PEA3 motifs in p
Neu664. Activation of p
Neu664 was virtually the same in MCF-7 and SK-BR-3 tumor cells, whereas a marked decrease in p
Neu250 activity was observed only in SK-BR-3 cells (Fig.
3), in complete contrast to their high c-erbB-2 expression (Fig.
2c). As discussed above, the striking overexpression of c-erbB-2 in SK-BR-3 cells can be explained by their multiple gene copies. In MCF-7 cells, the differential promoting activity of p
Neu250 could be explained by the relative increase in the transcription level compared with HEK-293 cells. In other aspects, in association with
c-erbB-2 gene amplification, up-regulation of transcriptional factors that control endogenous p
Neu remains possible. Conflicting results have been published on Ets regulation of c-erbB-2, with activation and repression of p
Neu by PEA3 factors having been reported [
46,
47]. The observation that Ets binding leads to a severe bend in DNA could be further support for our findings [
46]. When the number of PEA3 binding sites is reduced from four in p
Neu664 to one in p
Neu250, over-occupation of the single remaining site in p
Neu250 might hinder formation of the required DNA conformation rather than favor its reading.
The differential promoting activity of p
Neu664 and p
Neu250 in HEK-293 cells (Fig.
3) does not appear to be relevant to the transcriptional regulation of
c-erbB-2 because this gene is only weakly expressed in these cells (Fig.
1, lane 1). In contrast, HEK-293 cells continuously express ad5 E1A, which has been shown to target p
Neu [
48] as a repressor of HER2/
neu overexpression [
49], and has been proposed for use in cancer gene therapy [
50]. Like the Ets factors expressed in tumor cells, an equal level of E1A in HEK-293 cells might activate p
Neu664, which contains four PEA3 motifs, and repress p
Neu250, which has only one.
Our efforts to take advantage of natural cytotoxic anti-αGal antibodies as a means of destroying breast tumor cells, and to design a promoter specific for these undesirable cells, have to be considered as a preliminary contribution to the field of cancer gene therapy, given that our results have been obtained in cell line culture models. It has been shown that human primary breast tumors can be successfully engrafted into NOD/SCID mice and maintained in a growing state for more than 100 days [
51]. Moreover, αGalT(-/-) KO mice have been fortunately generated by others [
52,
53]. So to progress towards a gene therapy application, we are developing a two step procedure in mice. First, the distribution and expression of the transgene p
Neu250/mαGalT, cloned into the retroviral vector pcPMΔU3 (see Materials and methods), will be studied in a human breast cancer xenograft model. Various types of human breast tumor differentially expressing HER2/
neu will be implanted in NOD/SCID mice, and thereafter the transgene will be injected by a variety of methods. Second, αGalT-transduced tumor pieces will be transplanted into immunocompromised αGalT KO mice to evaluate the tumor destroying activity of purified human anti-αGal antibodies.