APOPTOTIC PROTEINS AND CANCER: MANY FACES

Faris Q Alenzi, Mohamed L. Salem, Fadel S. Alyaqoub, Fahad G. B. Alanazi, Wafa G. Alanazi, Mona G. Alanazi, Abdulmajeed G. Alanazi, Majed G. Alanazi, Hamdan Al- Shehri, Abdulwahab A Abuderman, Waleed Tamimi

Abstract


Complex genetic and epigenetic alterations that disrupt
the physiological regulation of apoptosis are thought to
be strategically critical for the carcinogenic process and
are thought to provide survival and growth advantages
for cancerous cells. Targeting and the induction of
apoptosis (programmed cell death) is an attractive target
for successful cytotoxic therapy for many different types
of cancer, including leukaemias and lymphomas.1,2
Bcl-2 protein
In normal liver cells, Bcl-2 family members have
essential roles in liver homeostasis. On other hand, in
carcinogenesis, these proteins play a significant role by
suppressing apoptotic death rather than stimulating cell
proliferation. The up-regulation of either Bcl-2 or BclxL in mouse liver has been shown to protect hepatocytes
from Fas-induced apoptosis and, therefore, liver
destruction in a dose-dependent manner.3 Recent data
from our work demonstrated the protein expression of
cytoplasmic Bcl-2 in 16% of chronic HCV patients with
no hepatocellular carcinoma (HCC) versus 8% in
patients with HCC.4 In HCC, Bcl-2 is significantly
down-regulated while Bcl-xL is predominately
expressed.5 However, Fiorentino et al reported
consistent increased level of Bcl-2 RNA in HCC which
may be suggest a post-transcription down-regulation of
Bcl-2 at the protein level.5
Another interesting finding was the delay of
liver tumour development in concordance with Bcl-2
up-regulation in TGFα/Bcl-2 double transgenic mice6
which appears to inhibit c-myc-induced liver
carcinogenesis7. However, the in vivo electrophoretic
transfer of Bcl-2 antisense oligonucleotide (ASO) into
liver demonstrated inhibitory effects on HCC in rat
models.8 The deferential expression of Bcl-2 with the
regards to tumours development could be contributed to
the expression status of p53. The expression of Bcl-2 is
significantly up-regulated in p53-positive HCC tissue
while down-regulated in p53-negative tissues.9
Changes in p53 and Bcl-2 protein expression
are a molecular hallmarks during hepatocarcinogenesis10
that occur in concordance with high expression of the
proliferating cell nuclear antigen (PCNA) and loss of
differentiation and HCC progression.11 The PCNA
expression is significantly elevated in late G1 and S
phases of proliferating cells, and has been used as a
biomarker for progression in different types of cancers
including HCC.12 The PCNA over-expression was even
considered as an indicator for increased risk of HCC
development in HCV-infected patients.13,14
Additionally, it was demonstrated that the cell
division rate and subsequently the size of thymocytes
population in vivo is significantly reduced by the
expression of Bcl-2 or Bcl-xL in some reports15 while
these two proteins can also inhibit apoptosis of dividing
cells16. Bradly et al demonstrated that over-expression
of Bax and Bcl-2 in T-cell of transgenic mice can result
in disturbing the cell cycle of dividing thymocytes. It
was found that while Bax has stimulatory effects, Bcl-2
has inhibitory effects on cell cycle of cycling
thymocytes. Furthermore, in activated T-cells, Bcl-2
overxpression was seen to delay the protein degradation
of the tumour suppressor gene p27, whereas Bax
accelerated that.17
Haemopoitic stem cells (HSC) with overexpressed Bcl-2 in Bcl-2-transgenic mice generated by
Demon et al were reported to remain viable after growth
factor withdrawal whereas HSC from WT mice did not
survive in the absence of growth factor. It was
demonstrated that HSC from Bcl-2-transgenic mice
responded to pro-growth factors (such as IL-1, IL-3, IL-
6, SCF and Flt3-ligand) with significantly faster and
more extensive proliferation with more delay in the cell
cycle entry when compared with that from WT mice.
Interestingly, when cultured with SCF, only 20% of WT
HSC remained viable after one week, while HSC from
Bcl-2-transgenic mice demonstrated greater survival
capabilities and more extensive proliferation. It was
concluded that over-expression of Bcl-2 and SCF/c-kit
signalling pathway are sufficient for HSC proliferation.
However, one should note that proliferation also
participated into the transformation of progenitor cells to
the myeloid lineage.18,19
P53, Fas and Apaf-1
A study by our group pointed out that in blast crisis
(BC) of chronic myeloid leukaemia (CML) the p53
expression is significantly increased when compared
with the chronic phase of CML.20 Interestingly, while
relatively high p53 expression was in general detected
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112 http://www.ayubmed.edu.pk/JAMC/24-1/Faris.pdf
along with up-regulation of apoptosis activating factor
(Apaf-1), a significant down-regulation of Apaf-1 was
oddly seen when p53 had become clearly over-expressed
suggesting a disturbance in the p53 pathway (11, CML
paper). Data from our group suggested decreased
expression level of p53 and Apaf-1 in patients with
BC.21 It seems that the Apaf-1 up-regulation by several
oncoproteins such as E2F1 is mechanistically critical for
facilitating the apoptosome assembly. Furthermore,
Kannan et al demonstrated the presence of point
mutation, deletions and other genomic rearrangements
of p53 gene in 25% of BC and that p53 is an upstream
regulator of Apaf-1.22 In fact, we previously suggested a
link between increased expression of p53, decreased
expression of Apaf-1 and lack of Fas expression in one
hand and progression of CML. Therefore, one should be
careful towards understanding the significant upregulation of these pro-apoptotic genes in BC
transformation as well as in response to therapeutic
approaches.
For any normally growing cell population
molecular defects either at gene level, mRNA level or
protein level for genes regulating proliferation and
apoptosis during cell cycle can interfere with the
balance between cell division and apoptosis for that
cellular population in vivo and provide strategically
advantageous scenario for carcinogenesis.23 Our
previous results indicates that apoptosis (via Fas-FasL)
play a role in regulating haemopoietic progenitor cell
kinetics in humans as it does in mice. It also showed that
caspases activation was required for the myeloid
maturation.24,25
Cell cycle proteins
Genes regulating apoptosis have significant impact on
the cell cycle. A number of studies demonstrated that
cell-cycle regulators could interconnect with
proliferation and apoptosis.
Both p16–/– and p21–/– mice are deficient in key
cell cycle genes, while lpr and gld mice (Fas and FasL
mutant mice, respectively) have a defective apoptotic
mechanism.24 However, Lewis et al26 showed that p16–/–
knockout mice have a higher self-replication capacity
than do wild-type (WT) mice, which links the cell cycle
and apoptosis. Similarly, p21–/– knockout mice have a
higher self-replication capacity (i.e., cell proliferation)
than do WT mice.
We showed that both lpr and gld mice have a
higher self-replication (i.e., cell proliferation) capacity
than do WT mice, which links apoptosis and
proliferation.24 Miyashita et al27 showed that the
restoration of p53 function resulted in down-regulation
of Bcl-2 levels and the occurrence of apoptosis. They
also showed that p53 activates the Bax promoter and
induces high levels of Bax mRNA and protein.
Moreover, Yin et al28 showed that Bax is required for
50% of p53-induced apoptosis. Gomez et al29
demonstrated a relationship between p27, cdk2 and
apoptosis in thymocytes, which was modulated by p53,
Bcl-2 and Bax. Thus, cdk2 activation seems to be the
key point at which the cell cycle and apoptosis meet.
Janicke et al30 showed that the retinoblastoma
(RB) gene is cleaved during apoptosis, at the caspase
consensus cleavage site (DEAD), resulting in a protein
product of 50 kDa. Dou et al31 showed that RB is also
cleaved on an interior site, producing proteins of 48 and
68 kDa. Fattman et al32 demonstrated that caspase-3 and
caspase-7 cleave RB at the DSID cleavage site, resulting
in proteins of 68 and 48 kDa.
These findings support a two-step model for
RB cleavage and a promoting role in chemotherapymediated apoptosis. Browne et al33 demonstrated that
RB is cleaved at the carboxyl terminal, producing 43-
and 30-kDa protein fragments. In addition, ZVAD was
found to inhibit the cleavage of RB, poly-ADP-ribose
polymerase (PARP) and apoptosis. In contrast, YVAD
did not inhibit primary carboxyl terminal cleavage of
RB and PARP. These results suggest that different
caspases are responsible for the cleavage of different
substrates during apoptosis.
In contrast, Suzuki and colleagues34
demonstrated that survivin interacts with cdk4, and, as a
result, p21 is released from its complex with cdk4 and
interacts with pro-caspase-3 in mitochondria, resulting
in inhibition of apoptosis. Cell-cycle transitions are
mediated through multiple phosphorylations of cyclincdk complexes. RB phosphorylation releases E2F
transcription factor, which activates certain genes during
S phase. Activation of p21 results in negative regulation
of the cell cycle. P21 interacts with cdk and PCNA,

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