RESUMO
Prothymosin alpha and parathymosin are two ubiquitous small acidic nuclear proteins that are thought to be involved in cell cycle progression, proliferation, and cell differentiation. In an effort to investigate the molecular function of the two proteins, we studied their spatial distribution by indirect immunofluorescence labeling and confocal scanning laser microscopy in relation to nuclear components involved in transcription, translation, and splicing. Results indicate that both proteins exhibit a punctuated nuclear distribution and are excluded by nucleoli. The distribution of prothymosin alpha in the nucleus is related to that of transcription sites, whereas the distribution of parathymosin correlates with early replication sites. This implies that prothymosin alpha and parathymosin are involved in transcription and replication, respectively. In addition to the punctate distribution, prothymosin alpha also is found concentrated in 1-6 nuclear domains per cell. These domains are found in more than 80% of randomly growing T24 human bladder carcinoma cells. They have a diameter of 0.2-2.5 microm, their size being inversely related to the number of domains per cell. The domains disappear during mitosis and the protein is excluded from the metaphase chromosomes. Double-labeling experiments associate these prothymosin alpha domains with PML and CstF64 containing nuclear bodies, but not with hnRNP-I containing domains or coiled bodies.
Assuntos
Núcleo Celular/metabolismo , Replicação do DNA , Precursores de Proteínas/metabolismo , RNA/biossíntese , Timosina/análogos & derivados , Núcleo Celular/genética , Humanos , Timosina/metabolismo , Células Tumorais CultivadasAssuntos
Núcleo Celular/química , Clorofórmio , Guanidinas , Fenóis , RNA/isolamento & purificação , Solventes , Tiocianatos , Células 3T3/química , Animais , Células HL-60/química , Humanos , Camundongos , Fenol , SoluçõesRESUMO
A number of studies have indicated that the small nuclear acidic protein prothymosin alpha is associated with cellular-proliferation events. For example, c-myc causes immediate transcriptional activation of prothymosin alpha, and prothymosin alpha antisense oligonucleotides inhibit myeloma cell division. To investigate the regulation of prothymosin alpha, we examined its mRNA and protein levels during the cell cycle of mononuclear cells and fibroblastic cells. We isolated immunoreactive material from cellular extracts and immunolocalized the protein to the nucleus during the cell cycle. We reported here that the material present in the cells is prothymosin alpha rather than the amino-terminal peptide thymosin alpha 1. [3H]Thymidine-incorporation studies associate maximum accumulation of mRNA and protein with the S/G2 phase of the cell cycle. This induction of prothymosin alpha mRNA seems to resemble cyclin B expression and is more pronounced in fibroblasts. Moreover, transient-transfection experiments indicate that transcription factor E2F is a strong positive regulator of the prothymosin alpha gene. Our results are consistent with the hypothesis that prothymosin alpha is involved in proliferation checkpoints of the cell cycle.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA , Precursores de Proteínas/biossíntese , Timosina/análogos & derivados , Animais , Sequência de Bases , Divisão Celular/fisiologia , Linhagem Celular , Meios de Cultura , Ciclinas , Fatores de Transcrição E2F , Fase G2 , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , RNA Mensageiro/análise , Proteína 1 de Ligação ao Retinoblastoma , Fase S , Timosina/biossíntese , Timosina/genética , Timosina/imunologia , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Expression of prothymosin alpha, an acidic nuclear protein implicated in cellular proliferation, has been reported to be regulated by c-myc in vitro. We have studied the correlation of expression levels between prothymosin alpha and c-myc, using three different in vivo systems, viz. normal ontogenic process of placental development, lytic viral infection and heat shock treatment. The two genes have been found to share a similar expression pattern, although prothymosin alpha mRNA remains always detectable, indicating the existence of yet another mechanism, in addition to c-myc, which regulates its expression in vivo.