It has been mentioned on several former occasions that in a few cases at least, the apparent deficiency in enzymic activity of neoplastic tissue could be caused by shortage of the required co-factors. They are listed in Figure 15 and like some of the metallic co-factors, to be discussed further on, are either loosely connected with the apoenzymes, acting as go-betweens, or are built into the protein, forming an integral part of the holoenzyme.

Fig. 15. Coenzymes or prosthetic groups and their function.

Of the coenzymes, involved in hydrogen (electron) transport, DPN and TPN were found by Weinhouse and collaborators140 to be reduced in primary and transplanted tumors of rat liver even in comparison with the non-neoplastic tissue adjacent to the DAB produced hepatomas. Similar observations were recorded by Sahasrabudhe et al.186 and Glock and McLean,104,105 whereby the Indian workers claimed that also the host tissues of tumor bearing animals (liver and spleen) showed low levels of pyridine nucleotides and the British team that TPN was particularly diminished. As the administration of niacinamide which normally stimulates the biosynthesis of pyridine nucleotides (see Kaplan et al.1**) did produce only an increase of the co-factors of 25-30% of that observed in the normal control animals, Sahasrabudhe221 suggested, as mentioned before, that this could be due to a preferential utilization of the adenine moiety for the synthesis of nucleic acids proceeding at a high rate in proliferating tumor tissues. This, of course, touches on the enzymatically controlled synthesis of the co-factors which, in the case of DPN, has also been discussed by Morton.188 He came to a different conclusion: in his view a low level of DPN pyro-phosphorylase (Fig. 16b), for instance in mouse mammary carcinoma and in mouse DAB hepatoma one-fifth of the corresponding normal tissues52, would account not only for the reduced content in coenzyme I but also for the high glycolytic activity of tumor cells. Thus, coupled with the proposal as formulated by Sahasrabudhe,221 with regard to a shortage in availability of adenine derivatives (ATP etc.83), the paucity of pyridine nucleotides alone could lead to imbalances of the energy producing metabolism in neoplastic tissue. Just as a lack of reduced DPN (DPNH) and pyridoxal phosphate, as quoted before,231 explained the apparently low activity of the composite 'cystine desulfurase' in tumors, so it was demonstrated by Shapiro et al.228 that diminished riboflavin (B2) or FAD levels in the mouse mammary carcinoma 755 did not necessarily reflect on the presence or absence of an enzyme containing this prosthetic group. Moreover the data of these authors 224-225,22a on pyri-doxine, thiamine, CoA and niacin, giving the impression of consistently low levels of these precursors of coenzymes in neoplastic tissue, indicate that some enzymic activities dependent on these co-factors may not be equally reduced.

Comparable with this situation where the levels of vitamins as nutritional forerunners of functional parts of holoenzymes could act as signposts of imbalances of enzyme systems, so the amounts of certain trace metals in tissues might disclose a normal or abnormal situation with regard to the enzymes requiring them. Bray and Harrap 58 have recently reviewed the role of specific metals in the action of enzymes and distinguished between those which as ions activate the biocatalysts in a loosely bound form (Ca, Fe, K, Mg, Mn, Na, Zn) and those in metallo-enzymes which are more firmly attached to the apo-enzyme and which are either dissociable (Ca, Fe, Mo) from some, or are non-dissociable (Fe, Mo, Zn) from others. Copper (Cu), cobalt (Co) and selenium (Se) have also been found in enzymes (tyrosinase, ascorbic acid oxidase, etc.), proteins (e.g. ceruloplasmin) and a vitamin (Bi2). Investigation as to the distributions of trace elements in normal and neoplastic tissues are, as yet, few and far between: Carruthers and Suntzeff 88 have reported on concentration changes of certain metals in mouse skin during the process of carcinogenesis induced by methylcholan-threne, and Olsen et al.191 have studied the content of normal and malignant human liver in Ag, Al, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sn, Zn. Some time ago, Coman et al. have pointed to the apparent deficiency of calcium in tumors, a deficiency which might reflect on the surface properties of the neoplastic cell, to be considered further on. As mentioned before, the author with Everett, Martin and Webb82 have started on a series of metal analyses (Cu, Mn, Mo and Zn, etc.) of rat livers of different age groups, some from animals fed on DAB. Again certain changes have been noted in trace element levels from young to older livers, and particularly with reference to Mo which showed a tendency towards low values in embryonic and in some hepatoma tissues. This, as stressed before, fits in very well with the correspondingly low levels of xanthine oxidase in such biological material. However further systematic work has to be carried out to obtain a clearer picture of the differences in trace metal content between healthy and neoplastic organs of a number of species. Samples derived from non-pathological human specimens of many parts of the body have been analysed by Tipton et a/.239 240 241 and Stitch.232 Their results could serve as a useful base line for further investigation in this field with particular reference to samples derived from tumors.

Fig. 16. Biosynthesis and breakdown of DPN.

6. Resume

Looking over the previous pages of this chapter, one can safely arrive at the conclusion that cells at least of certain tumors in certain species (mainly rodents) have on their wild and uncontrolled journey lost or even gained certain enzyme activities. It is only fair to state that a great number of references to contributions of workers with high reputation and skill, have not been quoted. The reason for this lies not in the bias of the writer in favor of proving a special point. It was simply caused by limitation of space and not by a process of bolstering up a preconceived idea. It is hoped that those distinguished workers who do not find their names in the list of references will understand this. Indeed, the writer is quite aware that the low or high level of specified enzymic activities have so far been described only in a limited number of primary or transplanted tumors, the majority in small laboratory animals. Taking into consideration the relative lack of information on human material; the question whether these changes refer to really rate determining catalysts which control 'bottle necks'; the undeniable existence of some contradictory results; the difficulty of comparing one set of data with another, because of different base line calculations; the complexity of subcellular organization with its distribution of enzymes between particles and solute; the complex interdependence of many metabolic pathways: these altered activities may be after all only a side issue and secondary symptoms of malignancy. On the other hand, if all observations made with widely different biocatalytic systems are put together, as it was done by Weber and Cantero 255 for the Novikoff hepatoma (see their Chart 1), an overall picture, perhaps approaching the state of affairs with many other tumors, emerges which is too meaningful to be solely the outcome of sheer coincidence. In nucleotide metabolism the catabolic events are abolished or diminished leading to an increased potential for nucleic acid synthesis and thus cell division; in the turnover of carbohydrates and derivatives, there exists a block against glycogen storage and glucose is utilized on the whole preferentially by glycolysis (but also by respiration) for energy production. This energy is not only used for the formation of nucleic acids but also of proteins, where the diminution in catabol-ism of amino-acids will help in the preservation of polypeptides. If all this together with a reduced production of lipids is amalgamated and linked with feedback or auto-catalytic mechanisms, the cancer cell in consequence appears to be characterized by biochemical properties which favor growth, multiplication and spreading at the cost of differentiation and a functional, quasi-social behaviour. Of course, changes in immunological and other cell surface properties, and increasing and fatal alterations in the relation between host tissues and mutated or virus-driven cell populations (hypertrophy → benign growths-→ neoplasia -→ anaplasia) must be taken into account. But perhaps in the future even these phenomena can be understood better in terms of their originating with enzymic changes which in turn reach back to EFS and DNA as superimposed controls.