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Nuclear phospholipase C β1, regulation of the cell cycle and progression of acute myeloid leukemia
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Advan. Enzyme Regul. 45 (2005) 126–135 www.elsevier.com/locate/advenzreg
Nuclear phospholipase C b1, regulation of the cell cycle and progression of acute myeloid leukemia Lucio Coccoa,, Lucia Manzolia, Giandomenico Palkab, Alberto M. Martellia,c,d a
Cellular Signaling Laboratory, Department of Anatomical Sciences, University of Bologna, Via Irnerio 48, Bologna 40126, Italy b Section of Genetics, Department of Biomedical Sciences, University of Chieti, Italy c ITOI-CNR, Bologna Unit, c/o IOR, Bologna, Italy d School of Pharmacy, University of Bologna, Italy
Introduction Late in the eighties and early in the nineties there appeared for the first time the evidence that polyphosphoinositides (PIs), even though as minor components in terms of amount, are present in the inner part of the cell nucleus and that their metabolism changes during cell growth and differentiation (Cocco et al., 1987, 1989; Divecha et al., 1991; Martelli et al., 1992). If we go back to the literature dealing with nuclear lipids, it seems that the nuclear PI story stems from the evidence obtained during the seventies and the early eighties, by Manzoli and co-workers. Indeed, they not only defined the composition of nuclear phospholipids but also made the first attempt to establish a possible metabolic role of these molecules by analyzing in vitro the relationship between nuclear phospholipids and DNA duplication, mRNA synthesis, and chromatin structural organization also in neoplastic cells (Cocco et al., 1985; Manzoli et al., 1978, 1977, 1974, 1972; Maraldi et al., 1984). Therefore, it is not Corresponding author. Tel.: +39 051 244467; fax: +39 051 251735.
E-mail address: [email protected] (L. Cocco). 0065-2571/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.advenzreg.2005.02.001
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surprising that having taken into account these data new investigators have focused their attention to the metabolic role of nuclear phospholipids and namely on the signaling activity exerted by all the players of the nuclear inositol lipid cycle. At present, the existence of a nuclear PI metabolism is well documented and recognized (Cocco et al., 2001b; Irvine, 2003). We do know that the nucleus is a functional compartment for PI metabolism (Martelli et al., 2003) and it is endowed with the enzymes involved in the classical PI cycle, such as kinases required for the synthesis of phosphatidylinositol (4,5)-bisphosphate (PIP2), phosphoinositidespecific phospholipase C (PLC), and diacylglycerol kinase (DGK) (Cocco et al., 2001a; D’Santos et al., 1998; Irvine, 2003; Martelli et al., 1999, 2000). Moreover, because of the role exerted in both cell growth and differentiation, there are clues that the nuclear PI metabolism could be implicated in neoplastic transformation (Martelli et al., 2004, 2002). Among the enzymes of the cycle nuclear PLC b1 appears to play a crucial role as a checkpoint in the G1 phase of the cell cycle (Faenza et al., 2000). Moreover, its activation and/or up-regulation is dependent on the control of type 1 insulin-like receptor (IGF-R) in both mouse fibroblasts and myboblasts (Faenza et al., 2004; Martelli et al., 1992), suggesting that its signalling activity is essential for the normal behavior of the cell, at least in culture. The recent discovery of a possible involvement of the deletion of PLC b1 gene in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) in humans (Lo Vasco et al., 2004) strengthens the contention that nuclear PLC signaling is essential for the physiological process such as cell growth and differentiation.
Nuclear PLC b1 and regulation of the cell cycle Up to now a large number of data links PLC to the nucleus, both evidencing the presence of various isoforms and the signaling activity (Cocco et al., 2001b, 2002). However, attention has been focused mainly on PLC b1, mainly because of the fact that this isoform is present in the nucleus as a resident enzyme. A possible activation of a nuclear PLC in quiescent Swiss 3T3 mouse fibroblasts stimulated to grow with insulin-like growth factor-1 (IGF-1), was reported in 1991 by a paper showing that IGF-1 produced in membrane-stripped nuclei a decrease in phosphatidylinositol (4,)phosphate (PIP) and PIP2 mass and a concomitant increase in diacylglycerol (DAG) levels, within 2 min stimulation (Divecha et al., 1991). This hinted at an activation of a nuclear PLC. On the contrary, no changes in PIP, PIP2, and DAG amount were detected in the cytoplasm or in nuclei in which the envelope was still present. Interestingly, bombesin, another powerful mitogen for these cells, stimulated inositide metabolism at the plasma membrane level (as evidenced by changes in the DAG mass measured in the cytoplasmatic fraction), but not in the nucleus. Concomitantly, our group demonstrated the presence of PLC b1 in nuclei of Swiss 3T3 mouse fibroblasts. The activity was up-regulated in response to IGF-1
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stimulation and was indeed responsible for the changes in the amount of both PIs and DAG in the nucleus (Martelli et al., 1992). PLC b1, when in the nucleus, plays an important role as a mediator of the mitogenic stimulus exerted by IGF-1 on Swiss 3T3 mouse fibroblasts. The inhibition of PLC b1 expression by antisense RNA makes these cells far less responsive to IGF-1, but not to platelet-derived growth factor (Manzoli et al., 1997). Nuclear PLC b1 is responsible for the increase in intranuclear DAG mass, which induces PKC-a to migrate to the nucleus (Cocco et al., 1996). The way in which PKC-a could affect normal cell proliferation has been pointed out by very recent findings showing that in NIH 3T3 mouse fibroblasts treated with powerful tumor promoter 12-myristate 13-acetate (PMA), PKC-a and PKC- activate the cyclin Dl and cyclin E promoters and thus markedly elevate the levels of both cyclin Dl and E. This results in higher proliferation rates. Upregulation of cylin D1 expression is mainly mediated through the AP-1 transcription factor enhancer element present in the cyclin D1 promoter (Soh and Weinstein, 2003). As concerned with regulation of the cell cycle, the downstream target of PLC b1 signaling is cyclin D3/cdk4 complex. Indeed, we have shown that PLC b1 when overexpressed in the nucleus activates the cyclin D3/cdk4 complex (but not cyclin E) and consequently the retinoblastoma tumor suppressor protein was hyperphosphorylated and finally the E2F-1 transcription factor was activated, giving rise to the progression through the G1 phase of the cell cycle (Faenza et al., 2000). Interestingly, we must take into account that stimulation of type 1 IGF-R activates nuclear inositide metabolism also in differentiation. Indeed differentiation of C2C12 myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLC b1. The timing of PLC b1 synthesis and its accumulation in the nucleus precedes that of the late muscle marker Troponin T by 24 h. The expression of a transfected PLC b1 mutant, lacking the nuclear localization signal acts as a dominant negative for nuclear translocation of PLC b1 and suppresses the differentiation of C2C12 myoblasts (Faenza et al., 2003). It has been proposed that myogenic factors regulate not only tissue-specific gene expression but also the exit from the cell cycle. At the onset of differentiation, MyoD activates cyclin D3 which then sequesters unphosphorylated retinoblastoma protein leading to irreversible exit of differentiating myoblasts from the cell cycle (Cenciarelli et al., 1999). We have also evidence that the activation of type 1 IGF-R in mouse C2C12 myoblasts, through upregulation of nuclear PLC b1, induces a significant increase of cyclin D3 expression, whilst forced expression of PLC b1 in the cytoplasm abolishes this effect (Cocco et al., manuscript in preparation). This fits with our previous observations showing that a downstream target of nuclear PLC b1 signalling is indeed the cyclin D3/cdk4 complex (Faenza et al., 2000) (Fig. 1).
Deletion of PLC b1 gene and progression from myelodysplastic syndrome to acute myeloid leukemia We have discussed in the previous chapter the fact that PLC b1 is a key intermediary in the signaling pathway which controls the normal progression or
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arrest in the G1 phase of the cell cycle. Common features are the dependency of this pathway on the occupancy of type 1 IGF-R and the downstream target, i.e., cyclin D3. This evidence hints at nuclear PLC b1 as a key signaling molecule in the normal behavior of the cell. We have recently characterized the human gene of PLC b1 (Peruzzi et al., 2002). The human PLC b1 gene, constituted of 36 small exons and introns, has been located on the short arm of human chromosome 20 (20pl2, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene (Peruzzi et al., 2000). It has been reported that aberrations in the chromosome 20 are present in MDS (Mori et al., 2000), but none of them appeared to be specific. Because of the availability of a specific probe for PLC b1, we moved our interest to the cytogenetic analysis of both AML, which is characterized by an uncontrolled proliferation of blasts, and of MDL, which can end up with AML (Zeidman et al., 1995). MDS constitutes a group of hematological disorders characterized by peripheral blood cytopenias, secondary to bone marrow dysfunction. It occurs predominantly in adult patients (usually 460 years of age) and evolves in AML in about 30% of the cases after variable intervals from diagnosis. The clinical transition is demonstrated by the clonal proliferation of the hematopoietic precursor that generates leukemic blasts unable to differentiate. It is considered that the evolution to AML is associated with additional genetic changes acquired by MDS patients. In addition AML evolving from MDS is much less responsive to chemotherapeutic agents than is de novo AML (Zeidman et al., 1995). Approximately 50% of MDS patients have a detectable chromosome abnormality, usually a total or partial deletion of chromosome 5 or 7 and/or trisomy 8, but translocations and amplifications are not very frequent. Allelic loss has been found on chromosomes 6q, 7p, 10p, 11q, 14q and 20q and even if there is no specific relationship between most of the rearrangements and the clinical outcome, MDS patients with abnormal karyotype are usually thought to be at higher risk for developing AML than MDS patients having normal karyotype (Bloomfield et al., 1997; Mrozek et al., 1997). It should be noted that the management of MDS patients showing normal karyotype by means of classic cytogenetic techniques is still a problem. It has recently been observed that the clinical follow-up of these patients is not sufficient since some of them have surprisingly worse and poorer clinical outcome than expected (Caligiuri and Bloomfield, 2000; Sheikhha et al., 2003). Therefore, it is still important to detect prognostic elements to add to the karyotype in MDS, in order to identify higher risk patients. The data by Lo Vasco et al. (2004) we are discussing now were obtained by analyzing two groups of patients. The first group is constituted of patients with hematological disorders and karyotype abnormalities revealed at GTG banding. Within this group five patients were chosen, showing at Spectral Karyotype (SKY) analysis, rearrangements involving the chromosome 20. Two patients, 80 and 77 years old, suffered respectively for MDS and refractory anemia with excess of blasts in transformation. These two patients did not receive therapy but only supportive care and died nine and six months, respectively, after the diagnosis. The other three patients affected by AML had cytosine-arabinoside treatment without acquiring the remission and died between 5 and 7 months after the diagnosis.
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The second group was constituted of patients diagnosed with AML (six patients) or MDS (nine patients). All these patients had standard GTG banding karyotype at diagnosis that was considered normal. MDS patients received supportive care, with no treatment excepting for those who developed AML. The AML affected patients were treated with standard protocol with chemotherapeutic drugs. In the first group of patients affected with different hematological disorders and complex karyotype for the presence of complex chromosome rearrangements and indecipherable markers, SKY analysis was performed for a more accurate identification of the karyotype which disclosed rearrangements of chromosome 20 consisting of total or partial gains or losses in five patients. In those five patients, SKY analysis disclosed 39 structural chromosome changes. All five patients had MDS at onset. In all five patients SKY disclosed chromosome aberrations consisting of simple or complex changes among which a translocation involving the long arm of chromosome 13 (13q) and the chromosome 20 [t(13;20)], observed in three of them, i.e., patients 1, 4 and 5. Using the specific probe for the PLC b1 gene, mapped on 20p12 (Peruzzi et al., 2000), fluorescence in situ hybridization (FISH) analysis disclosed the loss of one allele of the gene in all those three patients allowing the localization of the breakpoint on the short arm of chromosome 20. SKY showed respectively a t(11;20) with break point at band 11q23 in one patient and a t(17;20) with break at 17q11 in another one. Also in these two cases FISH analysis showed the monoallelic deletion of the PLC b1 gene allowing the localization of the breakpoint at band 20p12. Moreover, in patient 2 FISH analysis by using specific probes for CALM (OMIM *603025 located on 11q14) and MLL (OMIM *159555; located on 11q23) genes, frequently rearranged in AML karyotype (Calabrese et al., 1994; Castro et al., 1998), showed that the two genes were not rearranged because of the translocation. In the third patient, bearing the translocation between chromosomes 17 and 20, the deletion of PLC b1 affected both alleles. In addition, the immunocytochemical analysis on blast nuclei from a cytogenetic sample of this patient showed a complete lack of reactivity of the monoclonal antibody which recognizes PLC b1. There are reports showing that rearrangements of the short arm of chromosome 20 have been detected in a number of patients with solid tumors but rarely in hematological disorders (Gordon et al., 2003; Hu et al., 2003; Mori et al., 2000; Peng et al., 2002; Wada et al., 2002). In all the five patients the 20p rearrangement was associated with the deletion of the gene of PLC b1, which is a key signal transduction molecule in both cell differentiation and normal growth (Cocco et al., 2001a; Martelli et al., 1992). Nevertheless, the association with other chromosome aberrations hampers the definition of the role played by the 20p abnormalities in both the onset and the evolution of the disease. Therefore, the authors have focused their attention on the second group of patients with normal high resolution GTG banding karyotype. The FISH analysis with the probe for PLC b1 in six AML patients showed that two of them had a monoallelic deletion of the gene. These two patients died in a time frame ranging from 1 to 12 months after the diagnosis. The FISH analysis in nine patients affected with MDS, having normal high resolution
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GTG banding karyotype, showed that four of them had a monoallelic deletion of the gene. All of these patients died in a time frame ranging from 1 to 6 months after developing AML. The patient who died after 4 months after the diagnosis had secondary MDS after 4 years of therapy for Hodgkin’s disease; this is particularly interesting, as it is well known that secondary MDS are at high risk for developing leukemia. The total painting for chromosome 20 was normal in all the 15 analyzed patients. To establish the amplitude of the deletion and the possible involvement of genes other than PLC b1 within the 20pl2 region; PLC b1 has been used a probe for another gene localized in the same band, i.e., the PLC b4 gene, located at less than 1 Mb from PLC b1 (http://www.ensembl.org/Homo-sapiens/mapview?chr=20)). PLC b4 is another member of the PLC b family; in mammals it is specifically expressed in the nervous system and is involved in retinal phototransduction (Alvarez et al., 1995), whilst PLC b1 is mainly involved in the control of cell cycle (Faenza et al., 2000). Performing FISH analysis by using a cDNA probe for PLC b4, it was shown that five of the six patients bearing the monoallelic deletion of PLC b1 were normal, whilst one of them, patient 13, had also the monoallelic deletion of PLC b4. This result indicates that in this patient the region comprised between the two genes with a deletion spanning from 1 to 5 Mb (the resolution of standard high resolution karyotype) was lost. The results of the remaining five patients indicate that the absence of one allele of PLC b1 gene could be due to an interstitial deletion, which does not affect the PLC b4 gene located 0.1 Mb far from PLC b1. Immunocytochemical analysis on cytogenetic samples, using an anti PLC b1 antibody, showed that all the AML/MDS patients with normal results at FISH analysis also had normal staining of the nucleus, which is a preferential site for PLC b1 (Faenza et al., 2003). All the AML/MDS patients bearing the monoallelic deletion of PLC b1 gene showed reduced signal intensity when compared to normal control images using the same time of exposure. The clinical evolution and the progression of the disease of the MDS patients, all showing at diagnosis a normal karyotype and considered for this reason as a good prognosis, has been worse than expected, as all of them developed AML and died in a time frame ranging from 1 to 6 months. Moreover, the normal karyotype MDS patients, showing non-deletion of PLC b1 gene at FISH analysis, are still alive at least after 24 months after the diagnosis. All in all the genetic anomaly affecting a key signaling PLC, which is capable of controlling the cyclin D3/cdk4 checkpoint in G1 phase (Faenza et al., 2000), hints at a possible role in the pathophysiology of MDS and gives a first clue for the likelihood that PLC b1 could be involved in the progression of the disease (Fig. 1). Even though the data reported above must be considered as a preliminary observation based on a small group of patients, they constitute the rationale for the analysis of a large series of patients with a uniform clinical approach in order to prove that deletion of PLC b1 gene could provide a new tool to differentiate a high risk group within normal karyotype MDS patients, usually classified as at low risk for developing leukemia.
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L. Cocco et al. / Advan. Enzyme Regul. 45 (2005) 126–135 Type 1 IGF-R activation up-regulation
Chromosome 20p12 PLC β1 gene monoallelic deletion
NUCLEAR PLC β1 MDS (normal karyotype GTG banding)
targets Cyclin D3
AML
Myogenic differentiation Control of G1 phase progression
Fig. 1. Schematic diagram showing how nuclear PLC b1 is under the control of Type 1 IGF-R in normal cell growth and myogenic differentiation. It turns out that nuclear PLC b1 in both cases targets cyclin D3 and controls physiological cell behavior in the G1 phase. The monoallelic deletion of the gene, causing an imbalancement of the cell cycle, could be responsible for the progression of MDS to AML.
Summary A large number of observations have hinted at the fact that location impinges on function of some of the main players of nuclear inositol lipid cycle. PLC b1 is a wellknown example, given that it has been shown that only the enzyme located in the nucleus targets the cyclin D3/cdk4 complex, playing, in turn, a key role in the control of normal progression through the G1 phase of the cell cycle. The PLC b1 gene, which is constituted of 36 small exons and large introns, maps on the short arm of human chromosome 20 (20pl2, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene itself. The chromosome band 20pl2 has been shown to be rearranged in human diseases such as solid tumors without a more accurate definition of the alteration, maybe because of the absence of candidate genes or specific probes. Moreover, nonspecific alterations in chromosome 20 have been found in patients affected by MDS and acute myeloid leukemia AML. MDS is an adult hematological disease that evolves into AML in about 30% of the cases. The availability of a highly specific probe gave an opportunity to perform in patients affected with MDS/AML, associated with normal karyotype, painting and FISH analysis aimed to check the PLC b1 gene, given that this signaling molecule is a key player in the control of some checkpoints of the normal progression through the cell cycle. FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PLC b1 gene. In contrast, PLC b4, another gene coding for a signaling molecule, located on 20pl2.3 at a distance as far as less than 1 Mb from PLC b1, is unaffected in MDS patients with the deletion of PLC b1 gene,
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hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML, whilst the normal karyotype MDS patients, showing non-deletion of PLC b1 gene, are still alive at least 24 months after the diagnosis. The immunocytochemical analysis using an anti PLC b1monoclonal antibody showed that all the AML/MDS patients who were normal at FISH analysis also had normal staining of the nucleus, which is a preferential site for PLC b1. In contrast, the monoallelic deletion gave rise to a dramatic decrease of the nuclear staining suggesting a decreased expression of the nuclear PLC b1. The reported data strengthen the contention of a key role played by PLC b1 in the nucleus, suggest a possible involvement of PLC b1 in the progression of MDS to AML and pave the way for a larger investigation aimed at identifying a possible high risk group among MDS patients with a normal karyotype.
Acknowledgements This work was supported by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Italian ‘‘MIURCofin’’ and ‘‘MIUR-FIRB, and CARISBO Foundation, Bologna. Authors are grateful to A.M. Billi for the preparation of the manuscript.
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Nuclear phospholipase C b1, regulation of the cell cycle and progression of acute myeloid leukemia Lucio Coccoa,, Lucia Manzolia, Giandomenico Palkab, Alberto M. Martellia,c,d a
Cellular Signaling Laboratory, Department of Anatomical Sciences, University of Bologna, Via Irnerio 48, Bologna 40126, Italy b Section of Genetics, Department of Biomedical Sciences, University of Chieti, Italy c ITOI-CNR, Bologna Unit, c/o IOR, Bologna, Italy d School of Pharmacy, University of Bologna, Italy
Introduction Late in the eighties and early in the nineties there appeared for the first time the evidence that polyphosphoinositides (PIs), even though as minor components in terms of amount, are present in the inner part of the cell nucleus and that their metabolism changes during cell growth and differentiation (Cocco et al., 1987, 1989; Divecha et al., 1991; Martelli et al., 1992). If we go back to the literature dealing with nuclear lipids, it seems that the nuclear PI story stems from the evidence obtained during the seventies and the early eighties, by Manzoli and co-workers. Indeed, they not only defined the composition of nuclear phospholipids but also made the first attempt to establish a possible metabolic role of these molecules by analyzing in vitro the relationship between nuclear phospholipids and DNA duplication, mRNA synthesis, and chromatin structural organization also in neoplastic cells (Cocco et al., 1985; Manzoli et al., 1978, 1977, 1974, 1972; Maraldi et al., 1984). Therefore, it is not Corresponding author. Tel.: +39 051 244467; fax: +39 051 251735.
E-mail address: [email protected] (L. Cocco). 0065-2571/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.advenzreg.2005.02.001
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surprising that having taken into account these data new investigators have focused their attention to the metabolic role of nuclear phospholipids and namely on the signaling activity exerted by all the players of the nuclear inositol lipid cycle. At present, the existence of a nuclear PI metabolism is well documented and recognized (Cocco et al., 2001b; Irvine, 2003). We do know that the nucleus is a functional compartment for PI metabolism (Martelli et al., 2003) and it is endowed with the enzymes involved in the classical PI cycle, such as kinases required for the synthesis of phosphatidylinositol (4,5)-bisphosphate (PIP2), phosphoinositidespecific phospholipase C (PLC), and diacylglycerol kinase (DGK) (Cocco et al., 2001a; D’Santos et al., 1998; Irvine, 2003; Martelli et al., 1999, 2000). Moreover, because of the role exerted in both cell growth and differentiation, there are clues that the nuclear PI metabolism could be implicated in neoplastic transformation (Martelli et al., 2004, 2002). Among the enzymes of the cycle nuclear PLC b1 appears to play a crucial role as a checkpoint in the G1 phase of the cell cycle (Faenza et al., 2000). Moreover, its activation and/or up-regulation is dependent on the control of type 1 insulin-like receptor (IGF-R) in both mouse fibroblasts and myboblasts (Faenza et al., 2004; Martelli et al., 1992), suggesting that its signalling activity is essential for the normal behavior of the cell, at least in culture. The recent discovery of a possible involvement of the deletion of PLC b1 gene in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) in humans (Lo Vasco et al., 2004) strengthens the contention that nuclear PLC signaling is essential for the physiological process such as cell growth and differentiation.
Nuclear PLC b1 and regulation of the cell cycle Up to now a large number of data links PLC to the nucleus, both evidencing the presence of various isoforms and the signaling activity (Cocco et al., 2001b, 2002). However, attention has been focused mainly on PLC b1, mainly because of the fact that this isoform is present in the nucleus as a resident enzyme. A possible activation of a nuclear PLC in quiescent Swiss 3T3 mouse fibroblasts stimulated to grow with insulin-like growth factor-1 (IGF-1), was reported in 1991 by a paper showing that IGF-1 produced in membrane-stripped nuclei a decrease in phosphatidylinositol (4,)phosphate (PIP) and PIP2 mass and a concomitant increase in diacylglycerol (DAG) levels, within 2 min stimulation (Divecha et al., 1991). This hinted at an activation of a nuclear PLC. On the contrary, no changes in PIP, PIP2, and DAG amount were detected in the cytoplasm or in nuclei in which the envelope was still present. Interestingly, bombesin, another powerful mitogen for these cells, stimulated inositide metabolism at the plasma membrane level (as evidenced by changes in the DAG mass measured in the cytoplasmatic fraction), but not in the nucleus. Concomitantly, our group demonstrated the presence of PLC b1 in nuclei of Swiss 3T3 mouse fibroblasts. The activity was up-regulated in response to IGF-1
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stimulation and was indeed responsible for the changes in the amount of both PIs and DAG in the nucleus (Martelli et al., 1992). PLC b1, when in the nucleus, plays an important role as a mediator of the mitogenic stimulus exerted by IGF-1 on Swiss 3T3 mouse fibroblasts. The inhibition of PLC b1 expression by antisense RNA makes these cells far less responsive to IGF-1, but not to platelet-derived growth factor (Manzoli et al., 1997). Nuclear PLC b1 is responsible for the increase in intranuclear DAG mass, which induces PKC-a to migrate to the nucleus (Cocco et al., 1996). The way in which PKC-a could affect normal cell proliferation has been pointed out by very recent findings showing that in NIH 3T3 mouse fibroblasts treated with powerful tumor promoter 12-myristate 13-acetate (PMA), PKC-a and PKC- activate the cyclin Dl and cyclin E promoters and thus markedly elevate the levels of both cyclin Dl and E. This results in higher proliferation rates. Upregulation of cylin D1 expression is mainly mediated through the AP-1 transcription factor enhancer element present in the cyclin D1 promoter (Soh and Weinstein, 2003). As concerned with regulation of the cell cycle, the downstream target of PLC b1 signaling is cyclin D3/cdk4 complex. Indeed, we have shown that PLC b1 when overexpressed in the nucleus activates the cyclin D3/cdk4 complex (but not cyclin E) and consequently the retinoblastoma tumor suppressor protein was hyperphosphorylated and finally the E2F-1 transcription factor was activated, giving rise to the progression through the G1 phase of the cell cycle (Faenza et al., 2000). Interestingly, we must take into account that stimulation of type 1 IGF-R activates nuclear inositide metabolism also in differentiation. Indeed differentiation of C2C12 myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLC b1. The timing of PLC b1 synthesis and its accumulation in the nucleus precedes that of the late muscle marker Troponin T by 24 h. The expression of a transfected PLC b1 mutant, lacking the nuclear localization signal acts as a dominant negative for nuclear translocation of PLC b1 and suppresses the differentiation of C2C12 myoblasts (Faenza et al., 2003). It has been proposed that myogenic factors regulate not only tissue-specific gene expression but also the exit from the cell cycle. At the onset of differentiation, MyoD activates cyclin D3 which then sequesters unphosphorylated retinoblastoma protein leading to irreversible exit of differentiating myoblasts from the cell cycle (Cenciarelli et al., 1999). We have also evidence that the activation of type 1 IGF-R in mouse C2C12 myoblasts, through upregulation of nuclear PLC b1, induces a significant increase of cyclin D3 expression, whilst forced expression of PLC b1 in the cytoplasm abolishes this effect (Cocco et al., manuscript in preparation). This fits with our previous observations showing that a downstream target of nuclear PLC b1 signalling is indeed the cyclin D3/cdk4 complex (Faenza et al., 2000) (Fig. 1).
Deletion of PLC b1 gene and progression from myelodysplastic syndrome to acute myeloid leukemia We have discussed in the previous chapter the fact that PLC b1 is a key intermediary in the signaling pathway which controls the normal progression or
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arrest in the G1 phase of the cell cycle. Common features are the dependency of this pathway on the occupancy of type 1 IGF-R and the downstream target, i.e., cyclin D3. This evidence hints at nuclear PLC b1 as a key signaling molecule in the normal behavior of the cell. We have recently characterized the human gene of PLC b1 (Peruzzi et al., 2002). The human PLC b1 gene, constituted of 36 small exons and introns, has been located on the short arm of human chromosome 20 (20pl2, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene (Peruzzi et al., 2000). It has been reported that aberrations in the chromosome 20 are present in MDS (Mori et al., 2000), but none of them appeared to be specific. Because of the availability of a specific probe for PLC b1, we moved our interest to the cytogenetic analysis of both AML, which is characterized by an uncontrolled proliferation of blasts, and of MDL, which can end up with AML (Zeidman et al., 1995). MDS constitutes a group of hematological disorders characterized by peripheral blood cytopenias, secondary to bone marrow dysfunction. It occurs predominantly in adult patients (usually 460 years of age) and evolves in AML in about 30% of the cases after variable intervals from diagnosis. The clinical transition is demonstrated by the clonal proliferation of the hematopoietic precursor that generates leukemic blasts unable to differentiate. It is considered that the evolution to AML is associated with additional genetic changes acquired by MDS patients. In addition AML evolving from MDS is much less responsive to chemotherapeutic agents than is de novo AML (Zeidman et al., 1995). Approximately 50% of MDS patients have a detectable chromosome abnormality, usually a total or partial deletion of chromosome 5 or 7 and/or trisomy 8, but translocations and amplifications are not very frequent. Allelic loss has been found on chromosomes 6q, 7p, 10p, 11q, 14q and 20q and even if there is no specific relationship between most of the rearrangements and the clinical outcome, MDS patients with abnormal karyotype are usually thought to be at higher risk for developing AML than MDS patients having normal karyotype (Bloomfield et al., 1997; Mrozek et al., 1997). It should be noted that the management of MDS patients showing normal karyotype by means of classic cytogenetic techniques is still a problem. It has recently been observed that the clinical follow-up of these patients is not sufficient since some of them have surprisingly worse and poorer clinical outcome than expected (Caligiuri and Bloomfield, 2000; Sheikhha et al., 2003). Therefore, it is still important to detect prognostic elements to add to the karyotype in MDS, in order to identify higher risk patients. The data by Lo Vasco et al. (2004) we are discussing now were obtained by analyzing two groups of patients. The first group is constituted of patients with hematological disorders and karyotype abnormalities revealed at GTG banding. Within this group five patients were chosen, showing at Spectral Karyotype (SKY) analysis, rearrangements involving the chromosome 20. Two patients, 80 and 77 years old, suffered respectively for MDS and refractory anemia with excess of blasts in transformation. These two patients did not receive therapy but only supportive care and died nine and six months, respectively, after the diagnosis. The other three patients affected by AML had cytosine-arabinoside treatment without acquiring the remission and died between 5 and 7 months after the diagnosis.
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The second group was constituted of patients diagnosed with AML (six patients) or MDS (nine patients). All these patients had standard GTG banding karyotype at diagnosis that was considered normal. MDS patients received supportive care, with no treatment excepting for those who developed AML. The AML affected patients were treated with standard protocol with chemotherapeutic drugs. In the first group of patients affected with different hematological disorders and complex karyotype for the presence of complex chromosome rearrangements and indecipherable markers, SKY analysis was performed for a more accurate identification of the karyotype which disclosed rearrangements of chromosome 20 consisting of total or partial gains or losses in five patients. In those five patients, SKY analysis disclosed 39 structural chromosome changes. All five patients had MDS at onset. In all five patients SKY disclosed chromosome aberrations consisting of simple or complex changes among which a translocation involving the long arm of chromosome 13 (13q) and the chromosome 20 [t(13;20)], observed in three of them, i.e., patients 1, 4 and 5. Using the specific probe for the PLC b1 gene, mapped on 20p12 (Peruzzi et al., 2000), fluorescence in situ hybridization (FISH) analysis disclosed the loss of one allele of the gene in all those three patients allowing the localization of the breakpoint on the short arm of chromosome 20. SKY showed respectively a t(11;20) with break point at band 11q23 in one patient and a t(17;20) with break at 17q11 in another one. Also in these two cases FISH analysis showed the monoallelic deletion of the PLC b1 gene allowing the localization of the breakpoint at band 20p12. Moreover, in patient 2 FISH analysis by using specific probes for CALM (OMIM *603025 located on 11q14) and MLL (OMIM *159555; located on 11q23) genes, frequently rearranged in AML karyotype (Calabrese et al., 1994; Castro et al., 1998), showed that the two genes were not rearranged because of the translocation. In the third patient, bearing the translocation between chromosomes 17 and 20, the deletion of PLC b1 affected both alleles. In addition, the immunocytochemical analysis on blast nuclei from a cytogenetic sample of this patient showed a complete lack of reactivity of the monoclonal antibody which recognizes PLC b1. There are reports showing that rearrangements of the short arm of chromosome 20 have been detected in a number of patients with solid tumors but rarely in hematological disorders (Gordon et al., 2003; Hu et al., 2003; Mori et al., 2000; Peng et al., 2002; Wada et al., 2002). In all the five patients the 20p rearrangement was associated with the deletion of the gene of PLC b1, which is a key signal transduction molecule in both cell differentiation and normal growth (Cocco et al., 2001a; Martelli et al., 1992). Nevertheless, the association with other chromosome aberrations hampers the definition of the role played by the 20p abnormalities in both the onset and the evolution of the disease. Therefore, the authors have focused their attention on the second group of patients with normal high resolution GTG banding karyotype. The FISH analysis with the probe for PLC b1 in six AML patients showed that two of them had a monoallelic deletion of the gene. These two patients died in a time frame ranging from 1 to 12 months after the diagnosis. The FISH analysis in nine patients affected with MDS, having normal high resolution
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GTG banding karyotype, showed that four of them had a monoallelic deletion of the gene. All of these patients died in a time frame ranging from 1 to 6 months after developing AML. The patient who died after 4 months after the diagnosis had secondary MDS after 4 years of therapy for Hodgkin’s disease; this is particularly interesting, as it is well known that secondary MDS are at high risk for developing leukemia. The total painting for chromosome 20 was normal in all the 15 analyzed patients. To establish the amplitude of the deletion and the possible involvement of genes other than PLC b1 within the 20pl2 region; PLC b1 has been used a probe for another gene localized in the same band, i.e., the PLC b4 gene, located at less than 1 Mb from PLC b1 (http://www.ensembl.org/Homo-sapiens/mapview?chr=20)). PLC b4 is another member of the PLC b family; in mammals it is specifically expressed in the nervous system and is involved in retinal phototransduction (Alvarez et al., 1995), whilst PLC b1 is mainly involved in the control of cell cycle (Faenza et al., 2000). Performing FISH analysis by using a cDNA probe for PLC b4, it was shown that five of the six patients bearing the monoallelic deletion of PLC b1 were normal, whilst one of them, patient 13, had also the monoallelic deletion of PLC b4. This result indicates that in this patient the region comprised between the two genes with a deletion spanning from 1 to 5 Mb (the resolution of standard high resolution karyotype) was lost. The results of the remaining five patients indicate that the absence of one allele of PLC b1 gene could be due to an interstitial deletion, which does not affect the PLC b4 gene located 0.1 Mb far from PLC b1. Immunocytochemical analysis on cytogenetic samples, using an anti PLC b1 antibody, showed that all the AML/MDS patients with normal results at FISH analysis also had normal staining of the nucleus, which is a preferential site for PLC b1 (Faenza et al., 2003). All the AML/MDS patients bearing the monoallelic deletion of PLC b1 gene showed reduced signal intensity when compared to normal control images using the same time of exposure. The clinical evolution and the progression of the disease of the MDS patients, all showing at diagnosis a normal karyotype and considered for this reason as a good prognosis, has been worse than expected, as all of them developed AML and died in a time frame ranging from 1 to 6 months. Moreover, the normal karyotype MDS patients, showing non-deletion of PLC b1 gene at FISH analysis, are still alive at least after 24 months after the diagnosis. All in all the genetic anomaly affecting a key signaling PLC, which is capable of controlling the cyclin D3/cdk4 checkpoint in G1 phase (Faenza et al., 2000), hints at a possible role in the pathophysiology of MDS and gives a first clue for the likelihood that PLC b1 could be involved in the progression of the disease (Fig. 1). Even though the data reported above must be considered as a preliminary observation based on a small group of patients, they constitute the rationale for the analysis of a large series of patients with a uniform clinical approach in order to prove that deletion of PLC b1 gene could provide a new tool to differentiate a high risk group within normal karyotype MDS patients, usually classified as at low risk for developing leukemia.
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L. Cocco et al. / Advan. Enzyme Regul. 45 (2005) 126–135 Type 1 IGF-R activation up-regulation
Chromosome 20p12 PLC β1 gene monoallelic deletion
NUCLEAR PLC β1 MDS (normal karyotype GTG banding)
targets Cyclin D3
AML
Myogenic differentiation Control of G1 phase progression
Fig. 1. Schematic diagram showing how nuclear PLC b1 is under the control of Type 1 IGF-R in normal cell growth and myogenic differentiation. It turns out that nuclear PLC b1 in both cases targets cyclin D3 and controls physiological cell behavior in the G1 phase. The monoallelic deletion of the gene, causing an imbalancement of the cell cycle, could be responsible for the progression of MDS to AML.
Summary A large number of observations have hinted at the fact that location impinges on function of some of the main players of nuclear inositol lipid cycle. PLC b1 is a wellknown example, given that it has been shown that only the enzyme located in the nucleus targets the cyclin D3/cdk4 complex, playing, in turn, a key role in the control of normal progression through the G1 phase of the cell cycle. The PLC b1 gene, which is constituted of 36 small exons and large introns, maps on the short arm of human chromosome 20 (20pl2, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene itself. The chromosome band 20pl2 has been shown to be rearranged in human diseases such as solid tumors without a more accurate definition of the alteration, maybe because of the absence of candidate genes or specific probes. Moreover, nonspecific alterations in chromosome 20 have been found in patients affected by MDS and acute myeloid leukemia AML. MDS is an adult hematological disease that evolves into AML in about 30% of the cases. The availability of a highly specific probe gave an opportunity to perform in patients affected with MDS/AML, associated with normal karyotype, painting and FISH analysis aimed to check the PLC b1 gene, given that this signaling molecule is a key player in the control of some checkpoints of the normal progression through the cell cycle. FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PLC b1 gene. In contrast, PLC b4, another gene coding for a signaling molecule, located on 20pl2.3 at a distance as far as less than 1 Mb from PLC b1, is unaffected in MDS patients with the deletion of PLC b1 gene,
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hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML, whilst the normal karyotype MDS patients, showing non-deletion of PLC b1 gene, are still alive at least 24 months after the diagnosis. The immunocytochemical analysis using an anti PLC b1monoclonal antibody showed that all the AML/MDS patients who were normal at FISH analysis also had normal staining of the nucleus, which is a preferential site for PLC b1. In contrast, the monoallelic deletion gave rise to a dramatic decrease of the nuclear staining suggesting a decreased expression of the nuclear PLC b1. The reported data strengthen the contention of a key role played by PLC b1 in the nucleus, suggest a possible involvement of PLC b1 in the progression of MDS to AML and pave the way for a larger investigation aimed at identifying a possible high risk group among MDS patients with a normal karyotype.
Acknowledgements This work was supported by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Italian ‘‘MIURCofin’’ and ‘‘MIUR-FIRB, and CARISBO Foundation, Bologna. Authors are grateful to A.M. Billi for the preparation of the manuscript.
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