APOPTOTIC PROTEINS AND CANCER: MANY FACES

Abstract

Complex genetic and epigenetic alterations that disruptthe physiological regulation of apoptosis are thought tobe strategically critical for the carcinogenic process andare thought to provide survival and growth advantagesfor cancerous cells. Targeting and the induction ofapoptosis (programmed cell death) is an attractive targetfor successful cytotoxic therapy for many different typesof cancer, including leukaemias and lymphomas.1,2Bcl-2 proteinIn normal liver cells, Bcl-2 family members haveessential roles in liver homeostasis. On other hand, incarcinogenesis, these proteins play a significant role bysuppressing apoptotic death rather than stimulating cellproliferation. The up-regulation of either Bcl-2 or BclxL in mouse liver has been shown to protect hepatocytesfrom Fas-induced apoptosis and, therefore, liverdestruction in a dose-dependent manner.3 Recent datafrom our work demonstrated the protein expression ofcytoplasmic Bcl-2 in 16% of chronic HCV patients withno hepatocellular carcinoma (HCC) versus 8% inpatients with HCC.4 In HCC, Bcl-2 is significantlydown-regulated while Bcl-xL is predominatelyexpressed.5 However, Fiorentino et al reportedconsistent increased level of Bcl-2 RNA in HCC whichmay be suggest a post-transcription down-regulation ofBcl-2 at the protein level.5Another interesting finding was the delay ofliver tumour development in concordance with Bcl-2up-regulation in TGFα/Bcl-2 double transgenic mice6which appears to inhibit c-myc-induced livercarcinogenesis7. However, the in vivo electrophoretictransfer of Bcl-2 antisense oligonucleotide (ASO) intoliver demonstrated inhibitory effects on HCC in ratmodels.8 The deferential expression of Bcl-2 with theregards to tumours development could be contributed tothe expression status of p53. The expression of Bcl-2 issignificantly up-regulated in p53-positive HCC tissuewhile down-regulated in p53-negative tissues.9Changes in p53 and Bcl-2 protein expressionare a molecular hallmarks during hepatocarcinogenesis10that occur in concordance with high expression of theproliferating cell nuclear antigen (PCNA) and loss ofdifferentiation and HCC progression.11 The PCNAexpression is significantly elevated in late G1 and Sphases of proliferating cells, and has been used as abiomarker for progression in different types of cancersincluding HCC.12 The PCNA over-expression was evenconsidered as an indicator for increased risk of HCCdevelopment in HCV-infected patients.13,14Additionally, it was demonstrated that the celldivision rate and subsequently the size of thymocytespopulation in vivo is significantly reduced by theexpression of Bcl-2 or Bcl-xL in some reports15 whilethese two proteins can also inhibit apoptosis of dividingcells16. Bradly et al demonstrated that over-expressionof Bax and Bcl-2 in T-cell of transgenic mice can resultin disturbing the cell cycle of dividing thymocytes. Itwas found that while Bax has stimulatory effects, Bcl-2has inhibitory effects on cell cycle of cyclingthymocytes. Furthermore, in activated T-cells, Bcl-2overxpression was seen to delay the protein degradationof the tumour suppressor gene p27, whereas Baxaccelerated that.17Haemopoitic stem cells (HSC) with overexpressed Bcl-2 in Bcl-2-transgenic mice generated byDemon et al were reported to remain viable after growthfactor withdrawal whereas HSC from WT mice did notsurvive in the absence of growth factor. It wasdemonstrated that HSC from Bcl-2-transgenic miceresponded to pro-growth factors (such as IL-1, IL-3, IL-6, SCF and Flt3-ligand) with significantly faster andmore extensive proliferation with more delay in the cellcycle entry when compared with that from WT mice.Interestingly, when cultured with SCF, only 20% of WTHSC remained viable after one week, while HSC fromBcl-2-transgenic mice demonstrated greater survivalcapabilities and more extensive proliferation. It wasconcluded that over-expression of Bcl-2 and SCF/c-kitsignalling pathway are sufficient for HSC proliferation.However, one should note that proliferation alsoparticipated into the transformation of progenitor cells tothe myeloid lineage.18,19P53, Fas and Apaf-1A study by our group pointed out that in blast crisis(BC) of chronic myeloid leukaemia (CML) the p53expression is significantly increased when comparedwith the chronic phase of CML.20 Interestingly, whilerelatively high p53 expression was in general detectedJ Ayub Med Coll Abbottabad 2012;24(1)112 http://www.ayubmed.edu.pk/JAMC/24-1/Faris.pdfalong with up-regulation of apoptosis activating factor(Apaf-1), a significant down-regulation of Apaf-1 wasoddly seen when p53 had become clearly over-expressedsuggesting a disturbance in the p53 pathway (11, CMLpaper). Data from our group suggested decreasedexpression level of p53 and Apaf-1 in patients withBC.21 It seems that the Apaf-1 up-regulation by severaloncoproteins such as E2F1 is mechanistically critical forfacilitating the apoptosome assembly. Furthermore,Kannan et al demonstrated the presence of pointmutation, deletions and other genomic rearrangementsof p53 gene in 25% of BC and that p53 is an upstreamregulator of Apaf-1.22 In fact, we previously suggested alink between increased expression of p53, decreasedexpression of Apaf-1 and lack of Fas expression in onehand and progression of CML. Therefore, one should becareful towards understanding the significant upregulation of these pro-apoptotic genes in BCtransformation as well as in response to therapeuticapproaches.For any normally growing cell populationmolecular defects either at gene level, mRNA level orprotein level for genes regulating proliferation andapoptosis during cell cycle can interfere with thebalance between cell division and apoptosis for thatcellular population in vivo and provide strategicallyadvantageous scenario for carcinogenesis.23 Ourprevious results indicates that apoptosis (via Fas-FasL)play a role in regulating haemopoietic progenitor cellkinetics in humans as it does in mice. It also showed thatcaspases activation was required for the myeloidmaturation.24,25Cell cycle proteinsGenes regulating apoptosis have significant impact onthe cell cycle. A number of studies demonstrated thatcell-cycle regulators could interconnect withproliferation and apoptosis.Both p16–/– and p21–/– mice are deficient in keycell cycle genes, while lpr and gld mice (Fas and FasLmutant mice, respectively) have a defective apoptoticmechanism.24 However, Lewis et al26 showed that p16–/–knockout mice have a higher self-replication capacitythan do wild-type (WT) mice, which links the cell cycleand apoptosis. Similarly, p21–/– knockout mice have ahigher self-replication capacity (i.e., cell proliferation)than do WT mice.We showed that both lpr and gld mice have ahigher self-replication (i.e., cell proliferation) capacitythan do WT mice, which links apoptosis andproliferation.24 Miyashita et al27 showed that therestoration of p53 function resulted in down-regulationof Bcl-2 levels and the occurrence of apoptosis. Theyalso showed that p53 activates the Bax promoter andinduces high levels of Bax mRNA and protein.Moreover, Yin et al28 showed that Bax is required for50% of p53-induced apoptosis. Gomez et al29demonstrated a relationship between p27, cdk2 andapoptosis in thymocytes, which was modulated by p53,Bcl-2 and Bax. Thus, cdk2 activation seems to be thekey point at which the cell cycle and apoptosis meet.Janicke et al30 showed that the retinoblastoma(RB) gene is cleaved during apoptosis, at the caspaseconsensus cleavage site (DEAD), resulting in a proteinproduct of 50 kDa. Dou et al31 showed that RB is alsocleaved on an interior site, producing proteins of 48 and68 kDa. Fattman et al32 demonstrated that caspase-3 andcaspase-7 cleave RB at the DSID cleavage site, resultingin proteins of 68 and 48 kDa.These findings support a two-step model forRB cleavage and a promoting role in chemotherapymediated apoptosis. Browne et al33 demonstrated thatRB is cleaved at the carboxyl terminal, producing 43-and 30-kDa protein fragments. In addition, ZVAD wasfound to inhibit the cleavage of RB, poly-ADP-ribosepolymerase (PARP) and apoptosis. In contrast, YVADdid not inhibit primary carboxyl terminal cleavage ofRB and PARP. These results suggest that differentcaspases are responsible for the cleavage of differentsubstrates during apoptosis.In contrast, Suzuki and colleagues34demonstrated that survivin interacts with cdk4, and, as aresult, p21 is released from its complex with cdk4 andinteracts with pro-caspase-3 in mitochondria, resultingin inhibition of apoptosis. Cell-cycle transitions aremediated through multiple phosphorylations of cyclincdk complexes. RB phosphorylation releases E2Ftranscription factor, which activates certain genes duringS phase. Activation of p21 results in negative regulationof the cell cycle. P21 interacts with cdk and PCNA,