PubMed · 9453725
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J M Vignaud, A Duprez. 1997. [Cell division: practical approach].. https://pubmed.ncbi.nlm.nih.gov/9453725/
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5-Bromodeoxyuridine (BrdU) induces or suppresses senescence-associated genes in any types of mammalian cells. From a cDNA library upregulated by BrdU in HeLa cells, we identified the gene encoding VDUP1 as a senescence-associated gene in normal human fibroblasts. To address a role of VDUP1 in senescence, we established HeLa cell clones, V7 and V27, which express its mRNA in a doxycycline-dependent manner. Although their growth in liquid culture was moderately retarded, colony formation on semi-solid medium was strongly inhibited by overexpression of the mRNA. We also examined susceptibility of these clones to various reagents. Consequently, colony formation in liquid culture was strongly inhibited by paraquat in these clones. Their superoxide dismutase activity was normal.
The sequential organisation of replication foci during S phase in onion ( Allium cepa) and their relationship to the nuclear matrix were investigated. To discern their structural features and temporal firing sequence, immunodetection of 5-bromo-2'-deoxyuridine (BrdU) was carried out after in vivo feeding in synchronised cells released from a 14-h-long hydroxyurea block. Replication foci consisted of small replication granules, called replisomes, which clustered together. Analysis of synchronous binucleate cells that maintained in their two nuclei the specular symmetry of distribution of sister chromosomes in anaphase, showed that replication starts in small replication foci at the telomeric pole (pattern I), though the telomeres themselves formed large foci that were late-replicating. The rDNA replication foci (pattern II) also become replicated in early S phase. Replication of large foci, including the heterochromatin (IV), occurred in late S phase and finished at the centromeric nuclear pole (pattern V). Labelling of proliferating cell nuclear antigen (PCNA) in nuclear matrices, prepared from S-phase nuclei after extensive DNase digestion, demonstrated that replication foci were always stably anchored to the nuclear matrix. Thus, association with the nucleoskeleton is not exclusively mediated by the replicating or nascent DNA. The overlapping of patterns I, II and III in the nuclear matrix, in contrast to the results of BrdU localisation in nuclei, suggests that PCNA becomes associated with the nuclear matrix before the replication foci are operative, and remains bound during replication.
The epidermal growth factor (EGF) receptor is highly expressed in HaCaT keratinocytes as shown by Western blotting. Stimulation of HaCaT cells with EGF, and also with the serine protease thrombin, induced DNA synthesis, measured by incorporation of 5-bromo-2'-deoxyuridine into the DNA of proliferating cells. Using antibodies directed against the active form of the EGF receptor, we show that in HaCaT cells EGF and thrombin triggered a rapid activation of the EGF receptor, followed by the phosphorylation and activation of the extracellular signal-regulated protein kinase (ERK). Moreover, EGF and thrombin induced a transient synthesis of the zinc finger transcriptional regulator Egr-1. Proliferation, activation of ERK, and biosynthesis of Egr-1 was completely inhibited in EGF or thrombin-treated HaCaT cells by the MAP kinase kinase inhibitor PD98059 and by AG1487, an EGF receptor-specific tyrosine kinase inhibitor. These data indicate that phosphorylation and activation of both the EGF receptor and ERK are essential for mitogenic signaling via EGF and thrombin. The synthesis of Egr-1 in HaCaT cells as a result of EGF or thrombin stimulation suggests that Egr-1 may be an important "late" part of the EGF and thrombin-initiated signaling cascades. We postulate that Egr-1 may function as a "third messenger" in keratinocytes connecting mitogenic stimulation with changes in gene transcription.