Nucleotide sequence and cell cycle-associated differential expression of ZF5128, a novel Kruppel type zinc finger protein gene.

The nucleotide sequence of the ZF5128 gene, encoding a novel Kruppel type zinc finger protein, has been determined. The ZF5128 gene has a predicted 553-amino acid open reading frame, encoding a putative 61 kDa zinc finger protein. The N-terminus of the ZF5128 coding region has a well-conserved Kruppel-associated box (KRAB) domain that consists of KRAB box A and B, whereas the C-terminus contains a Kruppel type C2H2 zinc finger domain possessing nine C2H2 zinc finger motifs in tandem arrays with the highly conserved space region of the H/C-link. Each C2H2 zinc finger motif has a typical consensus sequence of CX2CX3FX5LX2HX3H. A 3.2 kb transcript specific for ZF5128 was expressed at high levels in the spleen, thymus, and peripheral blood leukocyte, and weakly expressed in the prostate, ovary, small intestine, colon (mucosal lining), placenta, lung, and pancreas. Although there was no detectable ZF5128 mRNA in unstimulated human peripheral T cells, it was first detectable 1.5 h after activation by anti-CD3 plus anti-CD28, and reached a maximum in 25-30 h. During the cell cycle progression of Jurkat T cells, the expression of ZF5128 mRNA appeared to be induced in G1 and reached a maximum in the S phase, but declined as the cells entered the G2/M phase. The 12-O-tetradecanoylphorbol 13-acetate-induced monocytic differentiation of U937, which also resulted in growth arrest, down-regulated the expression of ZF5128 mRNA. Taken together, these results indicate that ZF5128 is a novel gene encoding a Kruppel type C2H2 zinc finger protein and is regulated at the transcriptional level depending on tissue type and the cell cycle status to support cell proliferation.

Cloning and expression of human mitotic centromere-associated kinesin gene.

The human homologue of the hamster mitotic centromere-associated kinesin (HsMCAK) gene containing a central type motor domain was isolated from a Jurkat T-cell derived cDNA library. The HsMCAK gene has a predicted 723 amino acid open reading frame, encoding a 81 kDa protein that shares 79.2% homology with hamster MCAK. Unstimulated T lymphocytes contained no detectable HsMCAK-specific mRNA. Activation of resting T-cells by immobilized anti-CD3 resulted in the expression of a 2.9-kb transcript during the S phase of the cell cycle. The TPA-induced monocytic differentiation of U937 which also results in growth-arrest abruptly downregulates the expression of HsMCAK. Removal of TPA restored the growth of the cell through the retrodifferentiation process and the subsequent expression of HsMCAK. HsMCAK is expressed in tissues containing dividing cells, such as thymus, testis, small intestine, colon (mucosal lining), and placenta. These results suggest that the expression of HsMCAK is first detected in early S phase to support the proliferative response and is strictly regulated at the transcriptional level.

Expression of the apolipoprotein C-II gene during myelomonocytic differentiation of human leukemic cells.

Apolipoprotein C-II (apoC-II), which is known to activate lipoprotein lipase (LPL), was identified by ordered differential display (ODD)-polymerase chain reaction (PCR) as a cDNA fragment exhibiting a distinct increase in expression during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of promonocytic U937 cells into monocytes and macrophages. The amount of apoC-II mRNA expression detectable in U937 cells significantly increased and reached a maximum 24-48 h after treatment with 32 nM TPA. apoC-II mRNA was also detected in monocytic THP-1 cells but was not detected in promyelocytic HL-60 cells. In healthy human tissues, the most significant expression of apoC-II mRNA was in the liver. Although apoC-II mRNA expression was markedly up-regulated during the induced differentiation of HL-60 cells into monocytes and macrophages with 32 nM TPA, such expression was not induced during the differentiation of HL-60 cells into granulocytes with 1.25% dimethyl sulfoxide. These results suggest that human apoC-II expression is induced at the transcription level during myelomonocytic differentiation and may confer an important role to macrophages involved in normal lipid metabolism and atherosclerosis.

Nucleotide sequence and differential expression of the human 3-phosphoglycerate dehydrogenase gene.

The nucleotide sequence of Hs 3-PGDH gene, encoding human 3-phosphoglycerate dehydrogenase that catalyzes the initiating step in the phosphorylated pathway of serine biosynthesis, has been determined. The 3-PGDH gene has a predicted 533 amino acid open reading frame, encoding a 56.8kDa protein that shares 94.0% similarity with rat-liver 3-PGDH. Two different transcripts corresponding to 3-PGDH mRNA were detected in human normal tissues. A dominant 2.1kb transcript was expressed at high levels in prostate, testis, ovary, brain, liver, kidney, and pancreas, and weakly expressed in thymus, colon, and heart. A 710bp transcript also appeared as a weaker band where the 2.1kb mRNA was expressed, and it was more significant than the 2.1kb mRNA in heart and skeletal muscle. The TPA-induced monocytic differentiation of U937, which also resulted in growth arrest, abruptly downregulated the expression of 3-PGDH. Removal of TPA restored cell growth through the retrodifferentiation process and subsequent expression of 3-PGDH. The 3-PGDH mRNA was markedly expressed in human leukemias, lymphoma Sup-T1, colon adenocarcinoma COLO 320DM, epitheloid carcinoma HeLa S3, and murine lymphoma BW5147.G.1.4, but not in human leukemia K562. This report demonstrates that the human 3-PGDH gene is regulated at the transcriptional level depending on tissue specificty and cellular proliferative status, and its transcriptional regulation mechanism may be a useful target for diagnosis and therapy of cancer.

Chemopreventive agent resveratrol, a natural product derived from grapes, reversibly inhibits progression through S and G2 phases of the cell cycle in U937 cells.

Resveratrol, a natural product derived from grapes, has been shown to prevent carcinogenesis in murine models. We report here that resveratrol induces antiproliferation and arrests the S phase in human histiocytic lymphoma U937 cells. Resveratrol induces arrest in the S phase at low concentrations (30-60 microM), but high concentrations do not induce S phase accumulation in U937 cells. Removal of resveratrol from the culture medium stimulates U937 cells to reenter the cell cycle synchronously, as judged by the expression patterns of cyclin E, A and by fluorescent activated cell sorting analysis. These data demonstrate that resveratrol causes S phase arrest and reversible cell cycle arrest. Thus, resveratrol provides an important new cell cycle blocker as well as a cancer chemopreventive agent.

Isolation and characterization of a processed pseudogene for murine cyclin D3.

By using radiolabeled murine cyclin D3 cDNA as a probe, two cyclin D3 genomic clones, MCD3P-117 and MCD3P-327, were isolated from a murine genomic library constructed with murine liver DNA. Physical mapping and DNA sequence analysis revealed that these clones contain approximately 1.5 kb uninterrupted linear sequence similar to murine cyclin D3 cDNA, indicating that the 1.5 kb sequence is a processed pseudogene for cyclin D3. When the nucleotide sequence of the cyclin D3 pseudogene was compared with that of cyclin D3 cDNA at the nucleotide level, the pseudogene contained 229 bp of 5'- and 371 bp of 3'-untranslated regions, and a recognizable complete coding region that is 90% identical to murine cyclin D3. This sequence is bounded by the repeat sequence (GC/AGCTCTCC), which is common to many processed pseudogenes. However, multiple genetic lesions, including substitution, deletion and/or insertion events that result in modification of the reading frame were found in the pseudogene sequence. The pseudogene appeared to accumulate 67 random point mutations in the functional coding region composed of 879 nucleotide positions. It is thus estimated that the cyclin D3 pseudogene arose approximately 11 million years (Myr) ago. These data provide the first characterization of murine cyclin D3 pseudogene and insight into its evolutionary age.

Characterization of the murine cyclin D2 gene: exon/intron organization and promoter activity.

Cyclin D2 is normally expressed in G1 and promotes progression through G1 of the cell cycle. From a murine genomic library constructed with spleen DNA, two overlapping genomic clones of cyclin D2 were isolated. These clones contain most of the exon of cyclin D2 except exon 5. Characterization of these clones revealed that murine cyclin D2 mRNA spans over 18 kb and 5 exons ranging from 149 to approximately 462 bp in length, and suggested that exon 5 may be at least >5 kb downstream from exon 4. Primer extension analysis of cyclin D2 mRNA isolated from murine activated T cells detected 5 putative sites of transcription initiation. These are located at - 499, - 417, - 391, - 373, and - 349 relative to the translation start site, which is given as + 1. No consensus sequence for TATA box existed at an appropriate position within the promotor region. Instead, several putative transcriptional factor binding sites for C/EBP, PEA3, AP2, NF-Y, Sp1, c-Myc, GATA-1, AP1, v-Myb, and CREB were detected. The 5'-flanking region of the cyclin D2 gene up to nucleotide - 945 shared about 61% sequence homology between mouse and human. Functional analysis of promoter activity of the 5'-flanking region of cyclin D2 suggested that the region - 1,100 to - 805 including C/EBP, PEA3, AP2, NF-Y, c-Myc, and Sp1 may have a major positive regulatory activity for expression of cyclin D2.

The differential catalytic activity of alternatively spliced cdk2 alpha and cdk2 beta in the G1/S transition and early S phase.

Progression through the G1/S transition of the cell cycle is regulated by cyclin E/cdk2 and cyclin A/cdk2 complexes. We demonstrate that there are two forms of murine cdk2 (cdk2 alpha and beta). Cdk2 alpha consist of 298 amino acids, while cdk2 beta contains a 48-amino-acid insert between Met (196) and Val (197) of cdk2 alpha. Cdk2 beta results from differential splicing of the primary RNA transcript of the cdk2 gene. Although human cdk2 genomic DNA contained the sequence of the insert for the beta form, cdk2 beta was not detected by either Western blot or RT-PCR in human T-cells or several other human cell lines. Cdk2 beta expression in murine cells was similar to that of the phosphorylated, catalytically active form of cdk2 alpha. Cdk2 alpha and cdk2 beta have very similar binding activity to cyclin E and to the cdk inhibitor p27Kip1. The alternatively spliced cdk2 beta possesses catalytic activity in vivo and in vitro. The differential catalytic activity of these two forms of cdk2 suggests that cdk2 alpha and cdk2 beta may perform different functions at or near the G1/S transition and early S phase.