P34cdc2 Histone H1 Kinase Activity Research Articles

Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations.

The radiation response was investigated in two lymphoblastoid cell lines (LBC) derived from families with heterozygous germ-line missense mutations of p53 at codon 282 (LBC282) and 286 (LBC286), and compared to cells with wt/wt p53(LBC-N). By gel retardation assays, we show that p53-containing nuclear extracts from irradiated LBC282 and LBC286 markedly differ in their ability to bind to a p53 DNA consensus sequence, the former generating a shifted band whose intensity is 30-40% that of LBC-N, the latter generating an almost undetectable band. Unlike LBC286, which fail to arrest in G1 after irradiation, LBC282 have an apparently normal G1/S checkpoint, as they arrest in G1, like LBC-N. While in LBC-N, accumulation of p53 and transactivation of p21WAF1 increase rapidly and markedly by 3 h after exposure to gamma-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21WAF1 levels increase rapidly and significantly as in normal LBC. Apoptotic cells present 48 h after irradiation account for 32% in LBC-N, 8-9% in LBC282 and 5-7% in LBC286, while the dose of gamma-radiation required for killing 50% of cells (LD50) is 400 rads, 1190 rads and 3190 rads, respectively, hence indicating that the heterozygous mutations of p53 at codon 282 affects radioresistance and survival, but not the G1/S cell cycle control. In all LBC tested, radiation-induced apoptosis occurs in all phases of the cell cycle and appears not to directly involve changes in the levels of the apoptosis-associated proteins bcl-2, bax and mcl-1. Both basal as well as radiation-induced p53 and p21WAF1 proteins are detected by Western blotting of FACS-purified G1, S and G2/M fractions from the three cell lines. p34CDC2-Tyr15, the inactive form of p34CDC2 kinase phosphorylated on Tyr15, is found in S and G2/M fractions, but not in G1. However, 24 h after irradiation, its levels in these fractions diminish appreciably in LBC-N but not in the radioresistant LBC286 and LBC282. Concomitantly, p34CDC2 histone H1 kinase activity increases in the former, but not in the latter cell lines, hence suggesting a role for this protein in radiation-induced cell death. Altogether, this study shows that, in cells harbouring heterozygous mutations of p53, the G1 checkpoint is not necessarily disrupted, and this may be related to the endogenous p53 heterocomplexes having lost or not the capacity to bind DNA (and therefore transactivate target genes). Radiation-induced cell death is not cell cycle phase specific, does not involve the regulation of bcl-2, bax or mcl-1, but is associated with changes in the phosphorylation state and activation of p34CDC2 kinase.

Induction of A- and D-type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury

Hyperoxia causes a reproducible pattern of lung injury and repair in rodents, in which proliferation of alveolar epithelial cells (AEC) and fibroblasts is observed during recovery. We postulated that if quiescent cells are stimulated to reenter the cell cycle, then cyclin expression and cyclin-dependent protein kinase activity would be reactivated in AEC during the repair process after hyperoxic lung injury. To test this hypothesis, we exposed adult rats to short-term hyperoxia, followed by recovery for various times in room air. Cellular proliferation in vivo was confirmed by 1) flow cytometric analysis of DNA content (FACS) of freshly isolated AEC and 2) immunohistochemistry of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation into DNA on lung sections. The percentage of freshly isolated AEC in S phase and G2/M phase on FACS analysis increased twofold to a maximum of 16.5%, after 48 h in 100% oxygen and 48 h recovery in air. Cyclins A and D and p34cdc2 protein expression were also increased during the recovery period; while p33cdk2 and p34cdk4 increased only slightly. p34cdc2 histone H1 kinase activity, both in whole lung and in AEC, decreased initially after 48 h in oxygen. However, a marked increase in p34cdc2 kinase activity was observed at 48 h recovery in whole lung and returned to baseline by 72 h. In isolated and cultured AEC, p34cdc2 kinase activity was maximal at 24 h of recovery in air. We conclude that cyclins A and D and p34cdc2 protein expression and p34cdc2 kinase activity are increased in vivo during recovery from hyperoxic lung injury in both adult rat lungs and in AEC isolated from these lungs. We speculate that the induction of cyclin-dependent protein kinase activity is a key event in mediating the proliferative cellular repair response to lung injury.

Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts.

In serum-deprived human fibroblasts IMR-90 and WI-38 cells, the addition of fetal calf serum or basic fibroblast growth factor stimulates DNA synthesis in an extracellular Ca(2+)-dependent manner; the effect of serum on [3H]thymidine incorporation into DNA is 4-16-fold greater at 2.0 mM CaCl2 as compared with that at 0.03 mM CaCl2. By contrast, in SV40 virus-transformed WI-38 (SV-WI-38) cells DNA synthesis is essentially independent of the extracellular calcium concentration ([Ca]out) and serum growth factors. To explore the role of Ca2+ in mitogenic signal transduction through G1 to S phase cell cycle progression, we studied and compared the effect of [Ca]out on phosphorylation of RB protein, the product of a tumor suppressor retinoblastoma gene. In IMR-90 and WI-38 cells, serum or basic fibroblast growth factor induces an increase in the amount of hyperphosphorylated forms of RB protein in a manner strictly dependent on [Ca]out. In sharp contrast, in SV-WI-38 cells, the extent of RB phosphorylation is little affected by [Ca]out or the presence or absence of serum growth factors. In addition, potent calmodulin antagonists W-7 and calmidazolium, but not an inactive analogue W-12 or W-5, strongly inhibit serum-induced increases in DNA synthesis and RB phosphorylation in IMR-90 and WI-38 cells, whereas in SV-WI-38 cells, the inhibitory effect is much more limited. Under the same treatment conditions, we measured histone H1 kinase activity associated with anti-p34cdc2 immunoprecipitate and found that the serum-induced increase in p34cdc2 kinase activity is strongly dependent on [Ca]out and is potently inhibited by the active calmodulin antagonists in IMR-90 and WI-38 cells, but not in SV-WI-38 cells. In IMR-90 cells that have been incubated with serum in 0.03 mM [Ca]out for 24 h, restoration of [Ca]out to 2.0 mM results in initiation of DNA synthesis after 13 h and concomitant increases in RB phosphorylation and p34cdc2 histone H1 kinase activity. These results suggest that in human fibroblasts, Ca2+/calmodulin regulates the signaling cascade leading to cdc2 kinase activation, RB protein phosphorylation, and DNA synthesis and that this Ca(2+)-dependent regulation is abrogated in SV40-transformed cells.

Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.

Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.

Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner.

The regulation of p34cdc2 was investigated by overproducing p34cdc2, cyclin (A and B) and the wee1+ gene product (p107wee1) using a baculoviral expression system. p34cdc2 formed a functional complex with both cyclins as judged by co-precipitation, phosphorylation of cyclin in vitro, and activation of p34cdc2 histone H1 kinase activity. Co-production of p34cdc2 and p107wee1 in insect cells resulted in a minor population of p34cdc2 that was phosphorylated on tyrosine and displayed an altered electrophoretic mobility. When p34cdc2 and p107wee1 were co-produced with cyclin (A or B) in insect cells, there was a dramatic increase in the population of p34cdc2 that was phosphorylated on tyrosine and that displayed a shift in electrophoretic mobility. The phosphorylation of p34cdc2 on tyrosine was absolutely dependent upon the presence of kinase-active p107wee1. Tyrosine-specific as well as serine/threonine-specific protein kinase activities co-immunoprecipitated with p107wee1. These results suggest that cyclin functions to facilitate tyrosine phosphorylation of p34cdc2 and that p107wee1 functions to regulate p34cdc2, either directly or indirectly, by tyrosine phosphorylation.