John K. Chesters

Specificity and timing of the Zn2+ requirement for DNA synthesis by 3T3 cells

Addition of the chelator DTPA to synchronized cultures of 3T3 cells inhibited thymidine incorporation by up to 90% and only Zn2+ of the divalent cations tested was effective in reversing this effect. Fe2+ given alone had no effect on the inhibition by DTPA but when added to Zn2+ supplemented cultures increased thymidine incorporation from approximately 80-90 to over 100% of that in control cultures. Investigations indicated that the major requirement for Zn2+ was within the period from 8 h after stimulation of quiescent cells with serum until 3 h before the start of S phase. There was also an indication of a further requirement for Zn2+ at the G/S transition.

NATURE OF THE ZN2+ REQUIREMENT FOR DNA SYNTHESIS BY 3T3 CELLS

Transit of 3T3 cells from quiescence to S phase requires an adequate supply of Zn2+ during the second half of the transition. The nature of this requirement has been investigated. Completion of the Zn2(+)-dependent process required ongoing mRNA and protein synthesis but could be accomplished in serum-free medium. Combination of low Zn2+ availability with inhibition of mRNA synthesis by 5,6-dichlororibofuranosylbenzimidazole or of protein synthesis by cycloheximide resulted in the cells almost completely reverting to a quiescent state. The results suggest that Zn2+ is required for the accumulation and maintenance of a protein involved in the progression of untransformed cells into S phase.

A possible role for cyclins in the zinc requirements during G1 and G2 phases of the cell cycle.

Zinc has been shown to be required for the passage of cells through the mid-G1 phase of the cell cycle and for differentiation of myoblasts. However, it has been suggested that zinc has other roles during the cell cycle. The experiments reported here indicate that readily available zinc is not required for DNA synthesis per se but is needed for a process contemporaneous with the S phase and required for subsequent progress of the cells through G2 and mitosis. The G1 and S/G2 requirements for zinc showed virtually identical sensitivities to zinc deprivation. Each of the above requirements for zinc coincides with the induction of specific cyclin mRNAs, and the concentrations of these mRNAs have now been shown to decrease in the absence of adequate zinc. This is the first study to indicate a possible common factor underlying the requirement for available zinc during both cell replication and differentiation.

Zinc and the initiation of myoblast differentiation

Abstract Previous studies have indicated that a lack of available zinc inhibited myoblast differentiation as shown by a failure of the cells to fuse and low expression of creatine kinase mRNA and activity. However, the nature of the requirement for zinc and its relationship to the events leading to differentiation have been unclear. The current studies with C2C12 cells indicated that the muscle-specific enhancer present in the 5′-flanking region of the creatine kinase gene contributed to the zinc sensitivity of this enzyme. Because this enhancer can be activated by expression of the myogenic factors MyoD and myogenin, their sensitivity to zinc was investigated. The concentrations of both MyoD and, particularly, myogenin mRNA, were decreased by zinc deficiency. In vitro translation experiments suggested that these changes closely corresponded with alterations in their rates of synthesis. Further experiments failed to indicate a major effect of zinc on the stabilities of these mRNAs. Because an induction of myogenin mRNA is one of the earliest known events in myoblast differentiation, its particular sensitivity to lack of zinc suggests that zinc may be required before or during the initiation of myoblast differentiation.

Thymidine incorporation is less sensitive to lack of zinc in human than in rodent cells.

The inhibition of thymidine incorporation by inadequate availability of zinc induced by adding a chelator to the culture medium was significantly less in human cell lines than in rodent cell lines. In contrast, zinc uptake into the human cells was inhibited by the chelator to a greater extent than with rodent cells. The possible implications of these observations for the dietary zinc requirements of humans and rodents are discussed.

Effects of growth, food intake, and dietary zinc on diadenosine tetraphosphate concentrations in rats

The nucleotide diadenosine tetraphosphate has been suggested to function as a signal molecule for the initiation of DNA replication. Previous studies have indicated that diadenosine tetraphosphate is synthesized by certain aminoacyl tRNA synthetases and that diversion of AMP from the amino acid-enzyme complex to ATP to form diadenosine tetraphosphate is facilitated by zinc ions. The growth retardation of zinc-deficient rats is associated with specific reduction in DNA replication and also with a potentially growth-limiting decrease in food intake. The possibility has been investigated that in zinc-deficient rats, lack of Zn(2+) restricts diadenosine tetraphosphate synthesis, resulting in a failure to synthesize DNA and in a reduction in growth. The results indicate that the depressed growth potential caused by the reduction in food intake associated with the deficiency was sufficient to lower diadenosine tetraphosphate concentrations significantly in the liver and spleen. However, there was no indication of a specific effect of zinc deficiency on diadenosine tetraphosphate values.

5S Ribosomal RNA Synthesis in Zinc-Deficient Rats

Within a few days of being offered a Zn-deficient diet, young rats show an abrupt decline in growth rate and much effort has been directed to determining which function of Zn is most important in this context. Having reviewed the accumulated evidence, Chesters (1978) suggested that the critical requirement for Zn was associated with the alterations to the genetic expression of cells which occur during the induction of enzymes and the differentiation of tissues. However, low availability of Zn in EDTA-treated lymphocyte cultures resulted in delayed maturation of 32S ribosomal RNA precursor into 28S rRNA and reduced survival of 28S compared to 18S rRNA (Chesters, 1975). This sensitivity of post-transcriptional events to lack of Zn was hard to reconcile with the above hypothesis.