Cyclin A1 directly interacts with B-myb and cyclin A1/cdk2 phosphorylate B-myb at functionally important serine and threonine residues: tissue-specific regulation of B-myb function.

Cyclin A1 is tissue-specifically expressed during spermatogenesis, but it is also highly expressed in acute myeloid leukemia (AML). Its pathogenetic role in AML and in the cell cycle of leukemic blasts is unknown. B-myb is essential for G1/S transition and has been shown to be phosphorylated by the cyclin A2/cdk2 complex. Here it is demonstrated that cyclin A1 interacts with the C-terminal portion of B-myb as shown by glutathione S-transferase (GST) precipitation. This interaction is confined to cyclin A1 because binding could not be detected between cyclin A2 and B-myb. Also, cdk2 was not pulled down by GST-B-myb from U937 lysates. In addition, co-immunoprecipitation of cyclin A1 and B-myb in leukemic cells evidenced protein interaction in vivo. Baculovirus-expressed cyclin A1/cdk2 complexes were able to phosphorylate human as well as murine B-myb in vitro. Tryptic phosphopeptide mapping revealed that cyclin A1/cdk2 complexes phosphorylated the C-terminal part of B-myb at several sites including threonine 447, 490, and 497 and serine 581. These phosphorylation sites have been demonstrated to be important for the enhancement of B-myb transcriptional activity. Further studies showed that cyclin A1 cooperated with B-myb to transactivate myb binding site containing promoters including the promoter of the human cyclin A1 gene. Taken together, the data suggest that cyclin A1 is a tissue-specific regulator of B-myb function and activates B-myb in leukemic blasts. (Blood. 2001;97:2091-2097)

Cyclin E is the only cyclin-dependent kinase 2-associated cyclin that predicts metastasis and survival in early stage non-small cell lung cancer.

Progression through G1-S transition and S phase of the cell cycle is mediated by cyclin-dependent kinase 2 (cdk2), which interacts with several cyclins. Two of these, cyclin E and cyclin A2 (also known as cyclin A), are overexpressed in many cancers. Cyclin E2 and cyclin A1 are recently discovered cdk2-interacting cyclins that are found in malignant tumor cell lines and in acute myeloid leukemia, respectively. Expression and prognostic role of these cyclins in solid tumors is unknown. Here, we have analyzed expression and prognostic relevance of the cdk2-associated cyclins in non-small cell lung cancer (NSCLC). Fresh-frozen biopsies (n = 70) from completely resected tumors with stage I to IIIA NSCLC were studied. Gene expression was analyzed by quantitative real-time reverse transcription-PCR. Expression levels of cyclin E (P = 0.04) and cyclin A2 (P = 0.004) were significantly higher in the tumor samples than in normal controls. Cyclin A1, cyclin A2, and cyclin E2 expression levels did not have prognostic relevance for survival. The mean survival time associated with low and high levels of cyclin E was 69.4 and 47.2 months, respectively, which was statistically significant (P = 0.03). Differences in survival were particularly pronounced in stages I and II. Cyclin E was also closely associated with the development of distant metastasis (P = 0.01). Finally, we confirmed by immunohistochemistry analyses that cyclin E mRNA expression was closely associated with cyclin E protein expression. In conclusion, cyclin E is a strong independent prognostic indicator in patients with early-stage NSCLC, whereas cyclin E2, cyclin A1, and cyclin A2 do not have a prognostic role in NSCLC.

Methylation of the cyclin A1 promoter correlates with gene silencing in somatic cell lines, while tissue-specific expression of cyclin A1 is methylation independent.

Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.

c-myb transactivates the human cyclin A1 promoter and induces cyclin A1 gene expression.

Cyclin A1 differs from other cyclins in its highly restricted expression pattern. Besides its expression during spermatogenesis, cyclin A1 is also expressed in hematopoietic progenitor cells and in acute myeloid leukemia. We investigated mechanisms that might contribute to cyclin A1 expression in hematopoietic cells. Comparison of cyclin A1 and cyclin A promoter activity in adherent and myeloid leukemia cell lines showed that the cyclin A1 promoter is preferentially active in myeloid cell lines. This preferential activity was present in a small, 335-bp cyclin A1 promoter fragment that contained several potential c-myb binding sites. Coexpression of a c-myb expression vector with the cyclin A1 promoter constructs significantly increased the reporter activity in adherent CV-1 as well as in myeloid U937 cells. Gel-shift assays demonstrated that c-myb could bind to the cyclin A1 promoter at a binding site located near the transcription start site. Site-directed mutagenesis of this site decreased promoter transactivation by 50% in both KCL22 cells that express high levels of c-myb and in CV-1 cells that were transfected with c-myb. In addition, transfection of primary human embryonic fibroblasts with a c-myb expression vector led to induction of the endogenous cyclin A1 gene. Taken together, c-myb can directly transactivate the promoter of cyclin A1, and c-myb might be involved in the high-level expression of cyclin A1 observed in acute myeloid leukemia. These findings suggest that c-myb induces hematopoiesis-specific mechanisms of cell cycle regulation.

Cloning of the cyclin A1 genomic structure and characterization of the promoter region. GC boxes are essential for cell cycle-regulated transcription of the cyclin A1 gene.

Cyclin A1 is a recently cloned cyclin with high level expression in meiotic cells in the testis. However, it is also frequently expressed at high levels in acute myeloid leukemia. To elucidate the regulation of cyclin A1 gene expression, we cloned and analyzed the genomic structure of cyclin A1. It consists of 9 exons within 13 kilobase pairs. The TATA-less promoter initiates transcription from several start sites with the majority of transcripts beginning within a 4-base pair stretch. A construct containing a fragment from -190 to +145 showed the highest transcriptional activity. Transfection of cyclin A1 promoter constructs into S2 Drosophila cells demonstrated that Sp1 is essential for the activity of the promoter. Sp1, as well as Sp3, bound to four GC boxes between nucleotides -130 and -80 as observed by gel shift analysis. Mutations in two or more of the four GC boxes decreased promoter activity by >80%. The promoter was found to be cell cycle-regulated with highest activities found in late S and G2/M phase. Further analyses suggested that cell cycle regulation was accomplished by periodic repression of the GC boxes in G1 phase. Taken together, our data show that cyclin A1 promoter activity critically depends on four GC boxes, and members of the Sp1 family appear to be involved in directing expression of cyclin A1 in both a tissue- and cell cycle-specific manner.