A molecular basis for growth advantage provided to cancer cells by a tumor-specific enzyme: analysis of a very early event in carcinogenesis

A common feature of all cancer cells is their autonomous, uncontrolled proliferation. This autonomy is achieved by sequential mutations in and activation of several proliferation-promoting genes (oncogenes), and by the inactivation of several genes which inhibit proliferation (tumor-suppressor genes). Thus, the conversion of a normal into a cancerous cell is a multistage process, requiring the occurrence of many genetic (and biochemical) events in the same cell or its descendants. The pre-cancerous cells, the proliferation of which is not yet autonomous, must acquire some growth or selective advantages during the early stages of carcinogenesis in order to bring their population to such levels that will allow one or a few of them to undergo further events, and to proceed to autonomy and malignancy.
The enzymes responsible for such growth advantages may not belong to the oncogene or the tumor-suppressor gene families. Rather, they may be involved in metabolic pathways which facilitate and promote growth. A good example for such an enzyme is -glutamyl transpeptidase (GGT) which is induced to high levels in the very early stages of liver and skin cancer. The activity of GGT promotes carcinogenesis in that GGT-rich tumors grow three times faster than GGT-poor tumors. In our laboratory, we try to understand the molecular and biochemical basis for the advantage provided to tumor cells by GGT. GGT participates in several metabolic pathways. Each of them could possibly contribute to the selective growth advantage.
Many chemical carcinogens are also acutely toxic to cells, inhibiting their division or killing them. GGT participates in a metabolic pathway which detoxifies and excretes carcinogens, thus allowing GGT-rich pre-cancerous cells to survive in a carcinogen-induced toxic environment, whereas GGT-poor cells cease to proliferate or die out.
We have recently established that the abnormally high GGT activity in pre-cancerous cells induces the formation of reactive oxygen radicals and mutagenesis (1-5). Oxygen radicals cause DNA damage and are mutagenic, and have been implicated as functional in cancer promotion and in aging. GGT-induced oxidative damage may therefore help "initiated" pre-cancerous cells to undergo further genetic alterations that are required to attain autonomy.
Glutathione (GSH) is a small peptide that is important for detoxification. In addition, it helps DNA synthesis, the most important feature of proliferating cells, in that it facilitates the supply of DNA building blocks. GGT may participate in the elevation of glutathione concentration within cells, thus increasing their resistance to acute toxicity and facilitating their proliferation. We currently focus our studies on the role of GGT in glutathione synthesis. Our model systems are GGT-poor, non tumorigenic rat liver and mouse skin cells and their GGT-rich, tumorigenic counterparts.
As shown,GGT has two biochemical activities: hydrolysis of a -glutamyl bond and transpeptidation.
1. Glutathione + GGT ------> g-glutamyl-GGT + cysteinylglycine (hydrolysis)
2. cysteinylglycine ---> cysteine + glycine ---> transport
3. -glutamyl-GGT + cyst(e)ine ----------------> g-glutamylcyst(e)ine + GGT (transpeptidation)
In order to elucidate which of these activities is important for carcinogenesis, we have developed monoclonal antibodies that specifically inactivate either the hydrolytic (MAb 2A10) or the transpeptidatic (MAb 5F10) activities of GGT (6). We have shown that GSH levels in a GGT-rich, tumorigenic rat liver cell line (line M-22) are 2-3 fold higher than those in its GGT-poor, non tumorigenic parental line (line OC/CDE 22), and that GSH half life periods are 1.5-2 fold longer in the tumorigenic line. Under limiting concentrations of cysteine, resembling the in vivo conditions, the growth rate of GGT-rich cells is much higher than that of GGT-poor counterparts. We have studied the growth properties of these lines at limiting and non limiting cysteine in the presence and absence of MAb 5F10. This antibody inhibited the growth of M22 at limiting, but not at non limiting cysteine concentrations, whereas the growth of OC/CDE22 was only marginally affected. This indicates that transpeptidation is important for growth. We have also shown that mouse skin cell lines expressing moderate levels of GGT are less tumorigenic lines containing high GGT activity. We are currently studying the role of GGT in GSH biosynthesis and utilization in these lines. In addition, we have cloned human GGT cDNA in the sense and the antisense orientations under the control of the metallothionein promoter. GGT transfectants express high GGT activity levels in the presence but not in the absence of zinc. We are currently testing whether increase of GGT expression promotes tumorigenicity, and whether inhibition of GGT antisense expression abolishes or decreases tumorigenicity.

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