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A Novel, Extraneuronal Role for Cyclin-dependent Protein Kinase 5 (CDK5)

MODULATION OF cAMP-INDUCED APOPTOSIS IN RAT LEUKEMIA CELLS*
Open AccessPublished:March 21, 2002DOI:https://doi.org/10.1074/jbc.M112248200
      A number of cyclin-dependent protein kinase (CDK) inhibitors were tested for the ability to protect IPC-81 rat leukemic cells against cAMP-induced apoptosis. A near perfect proportionality was observed between inhibitor potency to protect against cAMP-induced apoptosis and to antagonize CDK5, and to a lesser extent, CDK2 and CDK1. Enforced expression of dominant negative CDK5 (but not CDK1-dn or CDK2-dn) protected against death, indicating that CDK5 activity was necessary for cAMP-induced apoptosis. The CDK inhibitors failed to protect the cells against daunorubicine-, staurosporine-, or okadaic acid-induced apoptosis. The inhibition of CDK5 prevented the cleavage of pro-caspase-3 in cAMP-treated cells. The cells could be saved closer to the moment of their onset of death by inhibitors of caspases than by inhibitors of CDK5. This suggested that the action of CDK5 was upstream of caspase activation. The cAMP treatment resulted in a moderate increase of the level of CDK5 mRNA and protein in IPC-81 wild-type cells. Such cAMP induction of CDK5 was not observed in cells expressing the inducible cAMP early repressor. The cAMP-induced increase of CDK5 contributed to apoptosis since cells overexpressing CDK5-wt were more sensitive for cAMP-induced death. These results demonstrate the first example of a proapoptotic CDK action upstream of caspase activation and of an extra-neuronal effect of CDK5.
      Cell death with the phenotype of apoptosis (for a review, see Refs.
      • Vaux D.L.
      • Strasser A.
      and
      • Strasser A.
      • O'Connor L.
      • Dixit V.M.
      ) can be triggered by cellular damage (“death by accident”), by withdrawal of survival factors (“death by neglect”), or by activation of pathways committed to death induction (“death by design”). Death by design can rely on preformed molecules (
      • Schneider P.
      • Tschopp J.
      ,
      • Walczak H.
      • Krammer P.H.
      ), such as members of the caspase family of proteases (
      • Nicholson D.W.
      ,
      • Wolf B.B.
      • Green D.R.
      ). This latter pathway can also be programmed in the sense that it requires ongoing gene transcription and protein translation (
      • Vaux D.L.
      • Strasser A.
      ,
      • Strasser A.
      • O'Connor L.
      • Dixit V.M.
      ).
      The rat promyelocytic IPC-81 cell line represents a unique cell system for the study of programmed cell death (
      • Gjertsen B.T.
      • Cressey L.I.
      • Ruchaud S.
      • Houge G.
      • Lanotte M.
      • Døskeland S.O.
      ,
      • Lanotte M.
      • Riviere J.B.
      • Hermouet S.
      • Houge G.
      • Vintermyr O.K.
      • Gjertsen B.T.
      • Døskeland S.O.
      ,
      • Ruchaud S.
      • Lanotte M.
      ). Cells start to undergo apoptosis within 3 h after activation of the cAMP-dependent protein kinase type I by physiological stimulators of adenylate cyclase-like prostaglandin E1 or by cAMP analogs, and the cells become apoptotic within 7 h (
      • Lanotte M.
      • Riviere J.B.
      • Hermouet S.
      • Houge G.
      • Vintermyr O.K.
      • Gjertsen B.T.
      • Døskeland S.O.
      ,
      • Gjertsen B.T.
      • Mellgren G.
      • Otten A.
      • Maronde E.
      • Genieser H.G.
      • Jastorff B.
      • Vintermyr O.K.
      • McKnight G.S.
      • Døskeland S.O.
      ,
      • Duprez E.
      • Gjertsen B.T.
      • Bernard O.
      • Lanotte M.
      • Døskeland S.O.
      ). IPC-81 cells with enforced expression of the cAMP-responsive element (CRE)
      The abbreviations used are: CRE
      cAMP-dependent protein kinase responsive element
      CREB
      cAMP-response element-binding protein
      CREM
      CRE modulator
      ICER
      inducible cAMP early repressor
      CDK
      cyclin-dependent protein kinase
      GSK-3 β
      glycogen-synthease kinase3
      8-CPT-cAMP
      8-(4-chlorophenylthio)-adenosine 3′: 5′-cyclic monophosphat
      zVAD-fmk
      z-val-ala-DL-asp-fluormethylketone
      dn
      dominant negative
      RCV
      roscovitine
      CHAPS
      (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)
      GFP
      green fluorescent protein
      EGFP
      enhanced GFP
      dn
      dominant-negative
      wt
      wild-type
      1The abbreviations used are: CRE
      cAMP-dependent protein kinase responsive element
      CREB
      cAMP-response element-binding protein
      CREM
      CRE modulator
      ICER
      inducible cAMP early repressor
      CDK
      cyclin-dependent protein kinase
      GSK-3 β
      glycogen-synthease kinase3
      8-CPT-cAMP
      8-(4-chlorophenylthio)-adenosine 3′: 5′-cyclic monophosphat
      zVAD-fmk
      z-val-ala-DL-asp-fluormethylketone
      dn
      dominant negative
      RCV
      roscovitine
      CHAPS
      (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)
      GFP
      green fluorescent protein
      EGFP
      enhanced GFP
      dn
      dominant-negative
      wt
      wild-type
      transcriptional blocker (ICER) (
      • Sassone-Corsi P.
      ) do not undergo apoptosis in response to cAMP (
      • Ruchaud S.
      • Seite P.
      • Foulkes N.S.
      • Sassone-Corsi P.
      • Lanotte M.
      ). This suggests that CRE-dependent gene transcription is essential for the cAMP-induced death. In a first approach to identify gene products involved in cAMP-induced cell death, a limited Atlas Array analysis was performed to compare cAMP-induced mRNA expression in IPC-81WT and IPC-81ICER cells. Among gene products already incriminated in apoptosis, only cyclin-dependent protein kinase 5 (CDK5) mRNA appeared to be selectively up-regulated in the wild-type cells (the present study). This observation spurred a closer study of the role of CDK5 in cAMP-induced cell death.
      The common general function of the CDK family members is to ensure the normal progression through the cell cycle, and they are tightly regulated by the sequential expression of cyclins (
      • van den Heuvel S.
      • Harlow E.
      ,
      • Arellano M.
      • Moreno S.
      ). Abnormal cell cycle control has been proposed to be a major mechanism for apoptotic cell death (
      • Ucker D.S.
      ,
      • Meikrantz W.
      • Schlegel R.
      ). The unscheduled activation of cell cycle-related CDKs such as CDK1 and CDK2 (
      • Chadebech P.
      • Truchet I.
      • Brichese L.
      • Valette A.
      ,
      • Harvey K.J.
      • Lukovic D.
      • Ucker D.S.
      ,
      • Hakem A.
      • Sasaki T.
      • Kozieradzki I.
      • Penninger J.M.
      ,
      • Meikrantz W.
      • Gisselbrecht S.
      • Tam S.W.
      • Schlegel R.
      ,
      • Meikrantz W.
      • Schlegel R.
      ,
      • Lu Y.
      • Tatsuka M.
      • Takebe H.
      • Yagi T.
      ) might have an impact late in apoptosis since they are activated by caspases (
      • Chadebech P.
      • Truchet I.
      • Brichese L.
      • Valette A.
      ,
      • Harvey K.J.
      • Lukovic D.
      • Ucker D.S.
      ).
      Cdk5 has high sequence similarity to the cell cycle regulating CDK family members, but it is neither activated by cyclins nor involved in cell cycle regulation ((
      • Meyerson M.
      • Enders G.H.
      • Wu C.L.
      • Su L.K.
      • Gorka C.
      • Nelson C.
      • Harlow E.
      • Tsai L.H.
      ,
      • Hellmich M.R.
      • Pant H.C.
      • Wada E.
      • Battey J.F.
      ); for a review, see Refs.
      • MacCioni R.B.
      and
      • MacCioni R.B.
      • Otth C.
      • Concha II,
      • Munoz J.P.
      ). Until recently, the expression of CDK5 and its activators, p35/25 and p39, were believed to be restricted to the nervous system (
      • Humbert S.
      • Dhavan R.
      • Tsai L.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness V.S., Jr.
      • Chae T.
      • Harlow E.
      ), where it contributes to neurite extension (
      • MacCioni R.B.
      • Otth C.
      • Concha II,
      • Munoz J.P.
      ,
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.
      • Tsai L.H.
      ). Cdk5 has been implicated in cell death during brain development (
      • Zhang Q.
      • Ahuja H.S.
      • Zakeri Z.F.
      • Wolgemuth D.J.
      ), in neurodegenerative diseases such as Alzheimer's dementia, Parkinson disease, and amyotrophic lateral sclerosis (
      • Lee M.S.
      • Kwon Y.T., Li, M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      ,
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ,
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      ,
      • Catania A.
      • Urban S.
      • Yan E.
      • Hao C.
      • Barron G.
      • Allalunis-Turner J.
      ,
      • Henchcliffe C.
      • Burke R.E.
      ,
      • Bajaj N.P.
      ), and in heat-shocked astrocytoma cells (
      • Gao C.
      • Negash S.
      • Wang H.S.
      • Ledee D.
      • Guo H.
      • Russell P.
      • Zelenka P.
      ).
      The evidence for a role of CDK5 outside the nervous system is scant. Both CDK5 and p35 have been detected in developing tissues during periods of programmed cell elimination (
      • Gao C.Y.
      • Zakeri Z.
      • Zhu Y., He, H.
      • Zelenka P.S.
      ,
      • Ahuja H.S.
      • Zhu Y.
      • Zakeri Z.
      ). Cdk5 has been shown by immunohistochemistry to be concentrated in dying cells (
      • Zhang Q.
      • Ahuja H.S.
      • Zakeri Z.F.
      • Wolgemuth D.J.
      ,
      • Ahuja H.S.
      • Zhu Y.
      • Zakeri Z.
      ) and in terminally differentiated cells (
      • Gao C.Y.
      • Zakeri Z.
      • Zhu Y., He, H.
      • Zelenka P.S.
      ).
      The present study demonstrates an essential role for CDK5 in the apoptotic process induced by the cAMP analog 8-(4-chlorophenylthio)-adenosine 3′: 5′-cyclic monophosphate (8-CPT-cAMP) in promyelocytic cells. By using cell-permeable CDK5 antagonists (
      • Meijer L.
      ), a requirement for CDK activity is demonstrated in a narrow time window preceding caspase activation. Finally, overexpression of CDK5-wt is shown to enhance death, whereas enforced expression of dominantly negative CDK5 will protect against apoptosis.

      DISCUSSION

      The cAMP-stimulated IPC-81 cells undergo unusually rapid programmed cell death, 50% apoptosis being observed within 4 to 5 h after the onset of cAMP challenge. The cAMP level needs to be elevated during the first 2 h, and only during this time can transcriptional inhibitors abrogate death (
      • Gjertsen B.T.
      • Cressey L.I.
      • Ruchaud S.
      • Houge G.
      • Lanotte M.
      • Døskeland S.O.
      ,
      • Ruchaud S.
      • Lanotte M.
      ). The present study shows that protein synthesis is required during the first 2.5 h of cAMP stimulation to achieve 50% death, whereas caspase activity is required for the first 3 h. By adding cell-permeable cyclin-dependent protein kinase inhibitors at various time points during the preapoptotic period, CDK activity was shown to be required prior to the caspase-dependent step,i.e. until ∼2.6 h after the onset of cAMP stimulation (Fig. 7). The complete recovery of cells co-incubated with CDK inhibitor and the agonistic cAMP analog 8-CPT-cAMP and then washed (Fig. 1H) argues that the CDK-dependent step was upstream of the irreversible part of the death execution pathway.
      So far, two mechanisms of action have been reported for inhibitors of cell cycle-related CDKs, such as CDK1 and CDK2, to protect against apoptotic cell death. The first is by arresting cycling cells (
      • van den Heuvel S.
      • Harlow E.
      ,
      • Meijer L.
      • Borgne A.
      • Mulner O.
      • Chong J.P.
      • Blow J.J.
      • Inagaki N.
      • Inagaki M.
      • Delcros J.G.
      • Moulinoux J.P.
      ) and thereby preventing them from entering parts of the cell cycle in which they are vulnerable to specific apoptogens (
      • Meikrantz W.
      • Schlegel R.
      ,
      • Darzynkiewicz Z.
      ,
      • Bruno S.
      • Ardelt B.
      • Skierski J.S.
      • Traganos F.
      • Darzynkiewicz Z.
      ,
      • Davis S.T.
      • Benson B.G.
      • Bramson H.N.
      • Chapman D.E.
      • Dickerson S.H.
      • Dold K.M.
      • Eberwein D.J.
      • Edelstein M.
      • Frye S.V.
      • Gampe Jr R.T.
      • Griffin R.J.
      • Harris P.A.
      • Hassell A.M.
      • Holmes W.D.
      • Hunter R.N.
      • Knick V.B.
      • Lackey K.
      • Lovejoy B.
      • Luzzio M.J.
      • Murray D.
      • Parker P.
      • Rocque W.J.
      • Shewchuk L.
      • Veal J.M.
      • Walker D.H.
      • Kuyper L.F.
      ). This appears to be an unlikely explanation for the protective effect of CDK inhibitors in the present study since they were efficient when given less than 2 h before cell death. During such a short period (<2h), only a small fraction of the cells can accumulate in any position of the cell cycle, making it unlikely that the CDK inhibitors protected IPC-81 cells through cell cycle arrest. Furthermore, IPC-81 cells pulsed with CDK inhibitor for 4 h showed enhanced, rather than inhibited, apoptosis in response to 8-CPT-cAMP (Fig. 4). This is opposite of what was expected if the CDK inhibitors acted to arrest cells in a less vulnerable part of the cell cycle.
      A second mechanism by which CDK inhibitors can protect against apoptosis is by blocking the unscheduled activity of CDK1 or CDK2 (
      • Harvey K.J.
      • Lukovic D.
      • Ucker D.S.
      ,
      • Hakem A.
      • Sasaki T.
      • Kozieradzki I.
      • Penninger J.M.
      ,
      • Meikrantz W.
      • Gisselbrecht S.
      • Tam S.W.
      • Schlegel R.
      ,
      • Meikrantz W.
      • Schlegel R.
      ,
      • Lu Y.
      • Tatsuka M.
      • Takebe H.
      • Yagi T.
      ,
      • Choi K.S.
      • Eom Y.W.
      • Kang Y.
      • Ha M.J.
      • Rhee H.
      • Yoon J.W.
      • Kim S.J.
      ). A number of apoptogens, including staurosporine and okadaic acid, can induce the unscheduled activation of CDK1 and CDK2, correlating with apoptosis (
      • Meikrantz W.
      • Gisselbrecht S.
      • Tam S.W.
      • Schlegel R.
      ). In IPC-81 cells, the CDK inhibitors were unable to protect against okadaic acid or staurosporine, even at concentrations above those required to protect against cAMP-induced death (Table II).
      It is noteworthy that the unscheduled activation of CDK1 (
      • Yao S.L.
      • McKenna K.A.
      • Sharkis S.J.
      • Bedi A.
      ) or CDK2 (
      • Harvey K.J.
      • Lukovic D.
      • Ucker D.S.
      ,
      • Kim S.G.
      • Kim S.N.
      • Jong H.S.
      • Kim N.K.
      • Hong S.H.
      • Kim S.J.
      • Bang Y.J.
      ,
      • Li W.
      • Fan J.
      • Bertino J.R.
      ,
      • Jin Y.H.
      • Yoo K.J.
      • Lee Y.H.
      • Lee S.K.
      ,
      • Gervais J.L.
      • Seth P.
      • Zhang H.
      ) appears to be downstream of caspase activation. One mechanism of caspase-dependent CDK activation is the cleavage of the CDK inhibitors p21Cip1/Waf1 and p27Kip1 (
      • Kim S.G.
      • Kim S.N.
      • Jong H.S.
      • Kim N.K.
      • Hong S.H.
      • Kim S.J.
      • Bang Y.J.
      ,
      • Jin Y.H.
      • Yoo K.J.
      • Lee Y.H.
      • Lee S.K.
      ,
      • Gervais J.L.
      • Seth P.
      • Zhang H.
      ). A schematic view of proposed links between CDK1, CDK2, and apoptosis is shown in Fig. 11A. However, this scheme could not explain the CDK involvement in IPC-81 cell death because CDK inhibition blocks 8-CPT-cAMP-induced cleavage of pro-caspase-3 and the CDK-dependent step occurs upstream of the caspase-dependent step. The introduction of dominant negative CDK1 or CDK2 (
      • van den Heuvel S.
      • Harlow E.
      ,
      • Arellano M.
      • Moreno S.
      ,
      • Harvey K.J.
      • Lukovic D.
      • Ucker D.S.
      ) failed to protect against 8-CPT-cAMP-induced cell death. The slow growth of these cells suggested that the level of CDK-dn was sufficient to perturb the cell cycle. We therefore concluded that CDK1 or CDK2 were unlikely to mediate the cAMP-induced IPC-81 cell death.
      Figure thumbnail gr11
      Figure 11Possible links between CDK5 and apoptosis.A shows a simplified scheme unifying numerous reports (19 and 63–67 and references therein) of caspase-dependent, unscheduled activation of CDK1 and CDK2 leading to apoptosis. A number of apoptogens, including staurosporine (1a), okadaic acid (1b), and tumor necrosis factor-α (1c), induce caspase activation (2) and activation of CDK1 or CDK2 (3), leading to apoptosis (5) via unknown routes (4).B shows the possible pathways involving CDK5 in cAMP-induced IPC-81 leukemic cell death. The increase of cellular cAMP (1) results in translocation of the catalytic subunit (C) of the cAMP-dependent kinase A to the nucleus (2) and the activation of CRE-governed gene transcription (3). This, in turn, leads to increased expression of CDK5 mRNA and protein (4 and 5) and possibly of CDK5 activator (x) or substrate (y). Caspase activation (7) and apoptosis (8) requires CDK5 phosphorylation of either the induced (y) protein substrate or of a preformed (s) protein substrate (6).
      Several findings pointed to the participation of CDK5 in the cAMP-induced cell death. A battery of CDK antagonists showed a near perfect correlation between the ability to protect against cAMP-induced apoptosis and to inhibit CDK5, and to a lesser extent, CDK2 and CDK1. Since CDK1 and CDK2 are unlikely mediators of the cell death, CDK5 is the most likely candidate. Secondly, dominant negative CDK5-T33 was able to protect against 8-CPT-cAMP-induced cell death. Thirdly, the fact that cAMP-induced death can occur in any phase of the cell cycle in the IPC-81 cell system (
      • Gjertsen B.T.
      • Cressey L.I.
      • Ruchaud S.
      • Houge G.
      • Lanotte M.
      • Døskeland S.O.
      ) favors the participation of a CDK family member, i.e. CDK5, which is not linked to the cell cycle (
      • Tang D.
      • Lee K.Y., Qi, Z.
      • Matsuura I.
      • Wang J.H.
      ).
      The involvement of CDK5 is further strengthened by the observation of up-regulation by 8-CPT-cAMP to death. The CDK5 mRNA level increased during the first 2 h of 8-CPT-cAMP stimulation, when cAMP-dependent gene transcription is critical for the cell death program to be initiated (
      • Lanotte M.
      • Riviere J.B.
      • Hermouet S.
      • Houge G.
      • Vintermyr O.K.
      • Gjertsen B.T.
      • Døskeland S.O.
      ,
      • Ruchaud S.
      • Seite P.
      • Foulkes N.S.
      • Sassone-Corsi P.
      • Lanotte M.
      ). Furthermore, the CDK5 protein level increased in the preapoptotic time window when protein synthesis was required for death. Cells expressing an inhibitor (ICER) of CRE-mediated gene transcription were blocked with respect to both up-regulation of CDK5 (Fig. 2) and death (
      • Ruchaud S.
      • Seite P.
      • Foulkes N.S.
      • Sassone-Corsi P.
      • Lanotte M.
      ). Finally, cells with enforced overexpression of CDK5 showed a more rapid apoptosis in response to 8-CPT-cAMP.
      Cdk5 is not the only cAMP-dependent factor required for IPC-81 cell death since overexpression of CDK5 to a level above that obtained in 8-CPT-cAMP-treated cells did not induce death by itself. The results of experiments where 8-CPT-cAMP was co-incubated with CDK5 inhibitor, which was subsequently removed, suggested that CDK5, in order to be proapoptotic, had to co-exist with other cAMP-induced factors. Such factors can be CDK5 activators that post-transcriptionally modify CDK5, CDK5 substrates, or enhancers of CDK5 substrate actions. Some of these options are depicted in Fig. 11B. We were unable to detect significant levels of the known CDK5 activator p35 in either untreated or 8-CPT-cAMP-treated IPC-81 cells, and the identity of the CDK5 activator(s) in IPC-81 cells is still unknown, as is the apoptosis-relevant substrate for CDK5. We do know, however, that the CDK-dependent step occurred about 25 min prior to the point when cells could no longer be protected by caspase inhibitors and that an additional couple of hours elapsed from the CDK-dependent commitment point until the cells became morphologically apoptotic with disarranged cytoskeleton.
      Hyperphosphorylation of the intermediate filament Tau with subsequent effects on the cytoskeleton has been described in cases when CDK5 has been activated (
      • MacCioni R.B.
      • Otth C.
      • Concha II,
      • Munoz J.P.
      ,
      • Lee M.S.
      • Kwon Y.T., Li, M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      ,
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      ,
      • Leclerc S.
      • Garnier M.
      • Hoessel R.
      • Marko D.
      • Bibb J.A.
      • Snyder G.L.
      • Greengard P.
      • Biernat J.
      • Wu Y.Z.
      • Mandelkow E.M.
      • Eisenbrand G.
      • Meijer L.
      ,
      • Zhang J.
      • Johnson G.V.
      ,
      • Munoz J.P.
      • Alvarez A.
      • MacCioni R.B.
      ). GSK-3 β has been described as an important factor in Tau hyperphosphorylation in Alzheimer-diseased neurons, and GSK-3 β is inhibited by a number of CDK antagonists (
      • Leclerc S.
      • Garnier M.
      • Hoessel R.
      • Marko D.
      • Bibb J.A.
      • Snyder G.L.
      • Greengard P.
      • Biernat J.
      • Wu Y.Z.
      • Mandelkow E.M.
      • Eisenbrand G.
      • Meijer L.
      ). However, GSK-3 β activity appeared not be required for cAMP-induced apoptosis in IPC-81 cells (Fig. 2B). Since CDK activity was nonessential during the last hours before cell death, it is unlikely that CDK5 acted by directly modulating cytoskeletal components, which probably were affected downstream of caspase activation.
      Although there is less precedence for a role of CDK5 than for CDK1 and CDK2 in apoptotic death, there is strong evidence of CDK5 involvement in neuronal and glioma cell death (
      • Kusakawa G.
      • Saito T.
      • Onuki R.
      • Ishiguro K.
      • Kishimoto T.
      • Hisanaga S.
      ,
      • Catania A.
      • Urban S.
      • Yan E.
      • Hao C.
      • Barron G.
      • Allalunis-Turner J.
      ,
      • Henchcliffe C.
      • Burke R.E.
      ,
      • Gao C.
      • Negash S.
      • Wang H.S.
      • Ledee D.
      • Guo H.
      • Russell P.
      • Zelenka P.
      ,
      • Zhang J.
      • Johnson G.V.
      ). None of these studies explored a relation between caspases and CDK5 action. Intriguing observations of increased CDK5 in dying cells outside the nervous system have led to the proposal of a role of CDK5 in extra-neuronal apoptosis (
      • Zhang Q.
      • Ahuja H.S.
      • Zakeri Z.F.
      • Wolgemuth D.J.
      ,
      • Ahuja H.S.
      • Zhu Y.
      • Zakeri Z.
      ,
      • Yin M.B.
      • Toth K.
      • Cao S.
      • Guo B.
      • Frank C.
      • Slocum H.K.
      • Rustum Y.M.
      ). The present study strongly supports this notion.
      In conclusion, cAMP-induced IPC-81 leukemia cell death appears to depend on CDK5 activity and is accompanied by CDK5 up-regulation mediated through CRE-dependent transcription. This may be the clearest example so far of an extra-neuronal effect of CDK5 and represents the first example of a proapoptotic CDK action upstream of caspase activation. This CDK-dependent programmed cell death system in IPC-81 cells offers a unique opportunity to identify apoptosis-associated CDK substrates upstream of caspase activation and to study CDK5 regulation outside the nervous system.

      ACKNOWLEDGEMENTS

      We are grateful to Dr. David Ucker for providing pCMV-CDK1-dn/CDK2-dn and for careful reading of the manuscript, Dr. Zahara Zakeri for providing pCMV-CDK5-T33, Dr. Laurent Meijer for providing important data on CDK inhibitor specificity prior to publication, Dr. John Eriksson for providing pcDNA-3.1-CDK5-wt, Dr. Ole Didrik Lærum and the Department of Pathology, Gades Institute, Haukeland Hospital for the excellent flow cytometry service, Dr. Ragnhild Ahlgren for valuable advice and participation during the preliminary phase of the investigation, and Dr. BjørnTore Gjertsen for valuable discussions. The excellent technical assistance of Nina Lied Larsen and Erna Finsås is highly appreciated.

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