We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, GO-phase cells, but increase dramatically (10-to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two-to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six-to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK-cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.

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The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- Thymidine kinase (TK) is an enzyme in the pyrimidine salvage pathway that catalyzes the phosphorylation of thymidine to dTMP. Many mammalian cells, including human HeLa cells, contain both cytoplasmic and mitochondrial forms of the enzyme (3), but the cytoplasmic form alone concerns us here. The regulation of the synthesis of TK is interesting because it is typical of that seen for many enzymes involved in DNA metabolism. TK activity is closely linked to the growth state of the cell, being present in rapidly growing but not in resting cells (13). In synchronized populations of cells, the activity is low in resting or Gl phase cells, but increases dramatically 10 to 20 h after the cells are released from arrest by serum stimulation, in parallel with the onset of DNA synthesis and entry into S phase. This induction is not absolutely dependent upon DNA synthesis (13), but does require both RNA and protein syntheses, suggesting that induction may be at the level of transcription. TK can also be induced by infection of resting cells with papovaviruses such as simian virus 40 (SV40) and polyoma (16,17), and the viral genes required for this induction are the large T antigens (30). Whether viral induction occurs by the same or a different mechanism(s) as serum induction is a question that remains to be answered.The TK gene provides a useful model system for carrying out a molecular analysis of genes that are cell cycle regulated and induced by viral infection. First, TK shows a great increase in activity (10-to 20-fold) after both serum and viral induction. Moreover, TK enzyme assays are both sensitive and easily performed. We can genetically select both for (hypoxanthine-aminopterin-thymidine media) and against (bromodeoxyuridine media) the TK+ phenotype, and many TK-cell lines exist. It has been shown that LTK-cells transfected to a TK+ phenotype with heterologous (human, rat, or hamster) chromosomal DNA containing a functional TK gene exhibit normal cell cycle regulation of the gene (31). Recent experiments with a cloned human gene (5) that the sequences required for cell cycle regulation are closely linked to the gene and function after transfection into TK-cells. Thus, this system should offer the chance to dissect the sequences involved in cell cycle-specific gene regulation.The mechanisms by which the expression of cell cycle-dependent genes is controlled, and by which the papovaviruses override these controls, remain obscure, although many of the initial observations were made more than 15 years ago. To a large extent this is because molecular probes for these genes and their transcripts have not been available. In this paper we report the isolation of both the human chromosomal TK locus and a functional human TK cDNA clone. Isolation of the chromosomal locus has previously been reported by several investigators (5,19,22), and our mapping is essentially in agreement with their data. In a...

Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the prepubertal gland and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution, and then increased again after weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double-labeling experiments in animals treated with E plus P for 3 d demonstrated that Stat5a was localized exclusively to cells containing both E and P receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland and suggest that these hormones act at the cellular level through their cognate receptors.

Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase that activates MAPK pathways, including the c-Jun NH 2 -terminal kinase (JNK) and p38 pathways. MLK3 and its family members have been implicated in JNK-mediated apoptosis. A survey of human cell lines revealed high levels of MLK3 in breast cancer cells. To learn more about MLK3 regulation and its signaling pathways in breast cancer cells, we engineered the estrogen-responsive human breast cancer cell line, MCF-7, to stably, inducibly express FLAG epitope-tagged MLK3. FLAG⅐MLK3 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by liquid chromatography/tandem mass spectrometry. Among the proteins identified were heat shock protein 90␣,␤ (Hsp90) and its kinase-specific cochaperone p50 cdc37 . We show that endogenous MLK3 complexes with Hsp90 and p50 cdc37 . Further experiments demonstrate that MLK3 associates with Hsp90/ p50 cdc37 through its catalytic domain in an activity-independent manner. Upon treatment of MCF-7 cells with geldanamycin, an ansamycin antibiotic that inhibits Hsp90 function, MLK3 levels decrease dramatically. Furthermore, tumor necrosis factor ␣-induced activation of MLK3 and JNK in MCF-7 cells is blocked by geldanamycin treatment. Our finding that geldanamycin treatment does not affect the cellular levels of the downstream signaling components, MAPK kinase 4, MAPK kinase 7, and JNK, suggests that Hsp90/p50 cdc37 regulates JNK signaling at the MAPK kinase kinase level. Previously identified Hsp90/p50 cdc37 clients include oncoprotein kinases and protein kinases that promote cellular proliferation and survival. Our findings reveal that Hsp90/p50 cdc37 also regulates protein kinases involved in apoptotic signaling.

We have identified a regulatory region in the human thymidine kinase gene promoter. A set of promoter deletion mutants was constructed, linked to the bacterial neomycin resistance gene, and stably transfected into Rat3 cells. It was shown that the region between 135 and 67 base pairs upstream of the cap site is required for conveying G1-S-phase regulation to the linked neo gene. In addition, primer extension assays demonstrated that the same transcriptional start sites were used in G1- and S-phase cells and in the various deletion mutants tested.

Signal transducer and activator of transcription (Stat)5a is a critical regulator of mammary gland development. Previous studies have focused on Stat5a's role in the late pregnant and lactating gland, and although active Stat5a is detectable in mammary epithelial cells in virgin mice, little is known about its role during early mammary gland development. In this report, we compare mammary gland morphology in pubertal and adult nulliparous wild-type and Stat5aϪ/Ϫ mice. The Stat5a-null mammary glands exhibited defects in secondary and side branching, providing evidence that Stat5a regulates these processes. In addition, Stat5aϪ/Ϫ mammary glands displayed an attenuated proliferative response to pregnancy levels of estrogen plus progesterone (EϩP), suggesting that it plays an important role in early pregnancy. Finally, we examined one potential mediator of Stat5a's effects, receptor activator of nuclear factor-B ligand (RANKL). Stat5aϪ/Ϫ mammary glands were defective in inducing RANKL in response to EϩP treatment. In addition, regulation of several reported RANKL targets, including inhibitor of DNA binding 2 (Id2), cyclin D1, and the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 , was altered in Stat5aϪ/Ϫ mammary cells, suggesting that one or more of these proteins mediate the effects of Stat5a in EϩP-treated mammary epithelial cells. (Endocrinology 151: 2876 -2885, 2010)

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizing segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. Computer analyses showed that the human DNA segments contained multiple homologies with sequences throughout the SV40 origin region, although sequences on the late side of the viral origin contained the strongest cross-hybridizing sequences. Because of the number and complexity of the matches detected, we could not determine unambiguously which of the many possible heteroduplexes between these DNAs was thermodynamically most favored. No hybridization of these human DNA sequences to any other segment of the SV40 genome was detected. In contrast, the human DNA segments isolated cross-hybridized with many sequences within the human genome. We tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence which enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientationindependent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. We propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an " activator" element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.