Role of the promoter in the regulation of the thymidine kinase gene.

To identify the regulatory elements of the human thymidine kinase (TK) gene, we have established stable cell lines carrying different chimeric constructs of the TK gene. Our results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G0 cells are stimulated by growth factors), TK mRNA levels are higher in G1-arrested cells than in proliferating cells; (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. We conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.     

Maintenance of dihydrofolate reductase enzyme after disappearance of DHFR mRNA during muscle cell differentiation

Summary Terminally differentiating mouse muscle cells were used to examine the relationship between mytogenic withdrawal from the cell cycle and the levels of dihydrofolate reductase (DHFR) mRNA and DHFR activity. Differentiation was induced by removal of fibroblast growth factor activity from the medium. DHFR mRNA was measured by a RNase protection assay. DHFR activity was measured by a spectrophotometric assay and by a [³H]methotrexate binding assay. Proliferative myoblasts contained four DHFR mRNA molecules and 1.8×10⁵ DHFR enzyme molecules. By 12.5 h after induction, when [³H]thymidine labeling indices showed all cells had withdrawn from the cell cycle, DHFR mRNA levels had declined to 0.7 copies per cell. In contrast, myogenic withdrawal did not result in reduced DHFR activity. Qualitatively similar results, i.e. down-regulation of mRNA and constitutive expression of activity, were observed in a methotrexate-selected muscle cell line with >50-fold amplification of the DHFR gene. Enzyme synthesis rate and stability measurements indicated that persistence of DHFR activity in postreplicative cells was due to a long enzyme lifetime rather than to continued synthesis from residual normal DHFR mRNA or an alternative mRNA species not detected by the RNase protection assay. Unlike DHFR, thymidine kinase (TK) activity disappeared rapidly as muscle cells differentiated. Both DHFR mRNA and TK mRNA are expressed in a replication-dependent manner; however, the enzymes encoded by these messages are subject to different fates in postreplicative cells. This work was supported by National Institutes of Health (Bethesda, MD) research grant GM34432, NIH Research Career Development Award AG00334, and a grant from the Medical Research Foundation of Oregon to G. F. M. E. E. S. was supported in part by Predoctoral Training Grant GM07774-08 from the Department of Health and Human Services, Washington, DC. and a N. L. Tartar Research Fellowship.     

Cell cycle regulation of thymidine kinase: residues near the carboxyl terminus are essential for the specific degradation of the enzyme at mitosis.

The level of human thymidine kinase (TK) polypeptide is subject to cell cycle regulation. The enzyme is barely detectable in G1 phase but increases 10- to 20-fold by M phase. The low level of human TK in G1 phase is due primarily to the specific degradation of the protein during cell division. Substitution of heterologous promoters, removal of the introns, and deletion of all of the 3' untranslated region from the human TK gene do not affect cell cycle regulation of the enzyme. However, deletion of the carboxyl-terminal 40 amino acids or fusion of beta-galactosidase to the carboxyl terminus of human TK completely abolishes cell cycle regulation and stabilizes the protein throughout the cell cycle. These alterations do not significantly alter the specific enzymatic activity of TK. Changing the carboxyl terminus or deletion of the last 10 amino acids does not alter cell cycle regulation. These data demonstrate that residues near the carboxyl terminus of TK are essential for the cell cycle phase-specific degradation of the enzyme.     

The promoter of the proliferating cell nuclear antigen (PCNA) gene is active in serum-derived cells.

mRNA levels for the Proliferating Cell Nuclear Antigen (PCNA) gene are growth regulated. PCNA promoters of different lengths were used to drive a linked reporter, the cDNA for human thymidine kinase (TK). After transfection into TK ts13 cells, stable cell lines were obtained. Regardless of promoter length, in all cell lines the levels of TK mRNA were roughly similar in serum-deprived and serum stimulated cells, confirming, by an independent method, that the growth regulation of PCNA mRNA levels doe not depend on the 5' flanking sequence of the PCNA gene.     

Cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in normal human cells.

The cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene was examined in normal human cells. Steady state RNA levels were monitored by Northern blot analysis using a plasmid (pChug 20.1) which contained the 1.3 kb GAPDH/UDG cDNA. The biosynthesis of the 37 kDa GAPDH/UDG protein was determined using an anti-human placental GAPDH/UDG monoclonal antibody to immunoprecipitate the radiolabeled protein. Increases in steady state GAPDH/UDG mRNA levels were cell cycle specific. A biphasic pattern was observed resulting in a 19-fold increase in the amount of GAPDH/UDG mRNA. The biosynthesis of the 37 kDa GAPDH/UDG protein displayed a similar biphasic regulation with a 7-fold increase. Pulse-chase experiments revealed a remarkably short half life of less than 1 hr. for the newly synthesized 37 kDa protein, comparable to that previously documented for a number of oncogenes. GAPDH/UDG mRNA levels were markedly reduced at 24 hr. when DNA synthesis was maximal. These results define the GAPDH/UDG gene as cell cycle regulated with a characteristic temporal sequence of expression in relation to DNA synthesis. The cell cycle synthesis of a labile 37 kDa monomer suggests a possible regulatory function for this multidimensional protein. Further, modulation of the GAPDH/UDG gene in the cell cycle may preclude its use as a reporter gene when the proliferative state of the cell is not kept constant.     

Structure and developmental expression of the chicken CDC2 kinase.

The cdc2 protein kinase plays a key role in controlling the eukaryotic cell cycle. We have isolated a cDNA clone for the chicken homolog of the cdc2 gene, raised antibodies against the corresponding protein, and studied the expression of cdc2 mRNA and protein during chicken embryonic development. The protein encoded by the chicken cdc2 cDNA shares extensive structural homology with cdc2 gene products from other species. Moreover, when expressed in fission yeast, the chicken cdc2 kinase is able to rescue a temperature-sensitive (ts) cdc2 mutant, demonstrating that it is functional as a cell cycle regulator. By Northern analysis and immunoblotting, we found that in total embryos both cdc2 mRNA and protein levels decreased substantially between day 3 and day 11 after egg laying, and no significant amounts of either cdc2 mRNA or protein were detected in adult liver, brain, heart or skeletal muscle. These data indicate the existence of a coarse correlation between the abundance of cdc2 mRNA and the proliferative state of a given tissue. Interestingly, however, when examining individual embryonic tissues, no correlation was observed between levels of cdc2 mRNA and protein, suggesting that cdc2 expression in developing chicken may be regulated at multiple levels.     

Regulation of creatine kinase induction in differentiating mouse myoblasts.

The regulation of creatine kinase (CK) induction during muscle differentiation was analyzed with MM14 mouse myoblasts. These cells withdraw from the cell cycle and commit to terminal differentiation when fed with mitogen-depleted medium. Myoblasts contained trace amounts of an isozyme of brain CK (designated BB-CK), but differentiation was accompanied by the induction of two other isozymes of muscle and brain CKs (designated MM-CK and MB-CK). Increased CK activity was detectable within 6 h of mitogen removal, 3 h after the first cells committed to differentiation and 6 h before fusion began. By 48 h, MM-CK activity increased more than 400-fold, MB-CK activity increased more than 150-fold, and BB-CK activity increased more than 10-fold. Antibodies prepared against purified mouse MM-CK cross-reacted with muscle and brain CKs (designated M-CK and B-CK, respectively) from a variety of species and were used to demonstrate that the increase in enzymatic activity was paralleled by an increase in the protein itself. CK antibodies were also used to aid in identifying cDNA clones to M-CK. cDNA sequences which corresponded to protein-coding regions cross-hybridized with B-CK mRNA; however, a subclone containing the 3'-nontranslated region was unique and was used to quantitate M-CK mRNA levels during myoblast differentiation. M-CK mRNA was not detectable in myoblasts, but within 5 to 6 h of mitogen withdrawal (6 to 7 h before fusion begins) it accumulated to about 30 molecules per cell. By 24 h, myotubes contained approximately 1,100 molecules per nucleus of M-CK mRNA.     

Metallothionein mRNA stability in chicken and mouse cells

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.     

Regulation of tropomyosin gene expression during myogenesis.

In skeletal muscle, tropomyosin has a critical role in transduction of calcium-induced contraction. Presently, little is known about the regulation of tropomyosin gene expression during myogenesis. In the present study, qualitative and quantitative changes in the nucleic acid populations of differentiating chicken embryo muscle cells in culture have been examined. Total nucleic acid content per nucleus increased about fivefold in fully developed myotubes as compared to mononucleated myoblasts. The contribution of deoxyribonucleic acid to the total nucleic acid population decreased from 24% in myoblasts to 5% of total nucleic acid in myotubes. Concomitant with the decrement in deoxyribonucleic acid contribution to total nucleic acid was an increase in polyadenylated ribonucleic acid (RNA) content per cell which reached levels in myotubes that were 17-fold higher than those of myoblasts. Specific changes in the RNA population during myogenesis were further investigated by quantitation of the synthetic capacity (messenger RNA levels) per cell for alpha- and beta-tropomyosin. Cell-free translation and immunoprecipitation demonstrated an approximately 40-fold increase in messenger RNA levels per nucleus for alpha- and beta-tropomyosin after fusion in the terminally differentiated myotubes. Indirect immunofluorescence with affinity-purified tropomyosin antibodies demonstrated the presence of tropomyosin-containing filaments in cells throughout myogenesis. Thus, the tropomyosin genes are constitutively expressed during muscle differentiation through the production of tropomyosin messenger RNA and translation into tropomyosin protein.     

The pattern of dihydrofolate reductase expression through the cell cycle in rodent and human cultured cells

We have examined the pattern of dihydrofolate reductase (DHFR) enzyme and mRNA levels in cell cycle stage-specific populations obtained by centrifugal elutriation in Chinese hamster ovary cells and in a derivative line in which the dihydrofolate reductase gene is amplified approximately 50-fold. On a per cell basis, we observed a 2-fold increase in DHFR activity as cells progressed from G1 to G2/M with a concomitant 2-fold increase in the rate of protein synthesis and steady state level of mRNA. Analysis of DHFR mRNA levels in cell cycle stage-specific mouse 3T6 and human 143 tk- cells gave a similar pattern. We also demonstrate that simple alterations in growth conditions prior to elutriations can dramatically increase the levels of DHFR mRNA in all cell cycle states, thereby indicating that growth response associated with the DHFR gene functions independent of the cell cycle. We conclude that during periods of exponential growth the increases in dihydrofolate reductase activity, rate of protein synthesis, and steady state levels of mRNA parallel the general increases in cell volume and protein content associated with normal progression through the cell cycle, and therefore DHFR cannot be considered a cell cycle-regulated enzyme.