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Regular Articles |
From the Department of Experimental Pathology,*
University of Ancona, Ancona; the Department of Human
Pathology,
University of Messina, Messina; the
Institute of Normal and Pathological
Cytomorphology,
National Research Council,
Chieti; and the Department of Oncology and
Neuroscience,
Clinical Pathology Section, G.
D’Annunzio University, Chieti, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methionine aminopeptidases (MetAPs) are
enzymes involved in the removal of the N-terminal methionine from
peptides and proteins. Two main MetAP isoforms have been identified so
far, precisely MetAP1 and MetAP2.5
The most significant
structural difference between the two is a large helical domain
insertion on the surface of the type 2 isozymes.6
MetAP2,
previously identified as an eukaryotic initiation factor-2-associated
67-kd protein, p67,7
regulates protein synthesis by
protecting the 
subunit of eukaryotic initiation factor-2 from
phosphorylation.8
MetAP2 expression correlates with cell
growth and nondividing cells do not show immunodetectable levels of
this enzyme.9
Moreover, MetAP2 is greatly induced by
phorbol myristate acetate.8
Recently, MetAP2 has been
identified as the molecular target of the angiostatic agents fumagillin
and ovalicin. These compounds selectively and covalently bind MetAP2
and block its aminopeptidase activity.9,10
Therefore,
MetAP2 might play a relevant role in cancer angiogenesis as well as in
the regulation of the proliferative activity of cells expressing this
enzyme.
Mesothelioma is an asbestos-related tumor that has been recently associated with the presence of SV40 DNA sequences.11 Prognosis is poor because of its marked resistance to conventional treatments, including surgical resection, chemotherapy, and radiotherapy, and the median survival of patients with this disease remains between 8 to 18 months. Mesothelioma cell growth is greatly influenced by the capability of these cells to release a number of growth factors including the pro-angiogenic fibroblast growth factor type 2, vascular endothelial growth factor (VEGF), and hepatocyte growth factor/scatter factor.12,13 In particular, it has been shown that mesothelioma cells express high VEGF, VEGF-C, and VEGF receptor mRNA levels14 and that elevated VEGF levels can be measured in pleural effusions of patients with mesothelioma.15 Moreover, we have also observed that VEGF potently stimulates mesothelioma cell proliferation by phosphorylation of the VEGF receptors, KDR and Flt-1, and that anti-VEGF antibodies inhibit the growth of these cells.16 Thus, angiogenic factors may promote mesothelioma growth not only by inducing tumor angiogenesis, but also by direct stimulation of cell proliferation. As a consequence, angiostatic molecules, capable of antagonizing growth factor actions, might have a considerable impact on mesothelioma cell survival.
Because MetAP2 is the intracellular target of anti-angiogenic agents and mesothelioma cells may be particularly sensitive to exposure to angiostatic molecules, we examined MetAP2 expression in mesothelioma cells, as well as the impact of this enzyme on cell survival and on some of the intracellular pathways regulating apoptosis. Here, we report that mesothelioma cells express higher MetAP2 mRNA levels compared to normal mesothelial cells and that MetAP2 inhibition induces mesothelioma cell apoptosis associated with a reduction in telomerase activity and bcl-2 down-regulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human primary mesothelial cells were established from patients and identified morphologically and by extensive phenotypic analysis as previously described.17 We used three different primary normal mesothelial cells, NM-2 and NM-3 (isolated from the peritoneal fluid of two distinct patients with nonneoplastic ascites) and NM-1 (obtained by pooling pleural effusion cells from four patients with congestive heart failure or liver disease). After 2 weeks in culture, 100% of the normal mesothelial cells stained positive for calretinin. They were expanded thereafter and used for the experiments between the third and seventh passage. Malignant mesothelial cell lines were established as previously described.18 Three distinct cell lines with different phenotype, biphasic (MM-B1), fibrosarcomatous (MM-F1), and epithelioid (HM) were used for the experiments between the eighth and twelfth passage. All mesothelial cells were maintained in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 1% L-glutamine, 1% penicillin-streptomycin (all from HyClone, Rome, Italy) at 37°C and 5% CO2.
Human umbilical vein endothelial cells (HUVECs) were obtained by Clonetics (San Diego, CA) and maintained in Opti-MEM medium (Life Technologies Inc., Milan, Italy) supplemented with 20% fetal calf serum. These cells were used between the second and fifth passage.
Proliferation Assay
Approximately 5 x 104 cells were exposed to oligonucleotides as described.19 Briefly, cells were washed three times with prewarmed (37°C), serum-free RPMI and incubated with either a MetAP2 anti-sense or a scrambled oligonucleotide previously mixed with 10 µg/ml of lipofectin (Life Technologies, Inc.) in serum-free RPMI. Cells were incubated for 4 hours at 37°C, 5% CO2. The medium was then removed and replaced with complete medium. Cells were collected after 1, 2, 3, and 4 days of incubation, stained with a 0.4% trypan blue solution (Sigma Chemical Co., Milan, Italy) and counted with a hemocytometer. All cell counts were done in triplicate. For analysis of DNA synthesis, 4 x 104 cells/ml were cultured for 24 hours in complete RPMI 1640 medium. Cells were then maintained in serum-free medium for 24 hours. This was replaced by complete medium containing the test agents and incubated for varying periods of time. At the end of the incubation, [3H]-thymidine (0.5 µCi/ml) was added for an additional 4 hours. Cells were then washed with phosphate-buffered saline (PBS)2+, incubated with ice-cold 5% trichloroacetic acid for 10 minutes, washed again with ethanol/ether (2:1) before being lysed with 0.5 ml of a solution containing PBS2+, 0.5% Triton, 200 mmol/L NH4OH, and 0.1% bovine serum albumin. Lysates were added to 3 ml of scintillation fluid and radioactivity was determined in a ß-scintillation analyzer (model Tri-Carb 2100TR; Packard, Milan, Italy) and counted for 1 minute.
Apoptosis
Apoptosis was assessed by DNA laddering and nucleosome formation. For DNA-laddering detection, DNA was extracted with phenol and subsequently precipitated with ethanol from cells treated from 1 to 4 days with either a MetAP2 anti-sense oligonucleotide or fumagillin in complete medium. Low-molecular weight DNA fragments were visualized by 2% agarose gel electrophoresis and ethidium bromide staining. Nucleosome formation was analyzed using a cell-death detection enzyme-linked immunosorbent assay (ELISA) kit (Roche Molecular Biochemicals, Milan, Italy). In experiments with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 (zVAD-fmk), the time of exposure to the MetAP2 anti-sense oligonucleotide was extended to 24 hours to obtain a reduction of the MetAP2 protein level before the addition of the caspase inhibitor.
Morphology
Cells suspensions were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.2, for 30 minutes, rinsed three times with the same buffer, postfixed with 1% OsO4, dehydrated with increasing concentrations of ethanol and embedded in the Epon 812 resin. Semi-thin sections were prepared using an appropriate microtome, stained with 1% toluidine blue at 40°C and examined by light microscopy. Ultra-thin sections, collected on 200-mesh copper grids, were stained with uranyl acetate and lead citrate and examined with a Zeiss 109 electron microscope at 80 kV.
Northern Blotting
Total RNA was extracted from subconfluent cells using the Trizol reagent (Life Technologies). RNA (15 µg) was separated on a 1% agarose/formaldehyde gel, transferred to a Hybond N+ nylon membrane overnight, and UV-cross-linked (Stratalinker 2400; Stratagene, Milan, Italy). The MetAP1, MetAP2, bcl-2, and bax probes were synthesized from full-length human cDNA by random primed 32P-labeling. Blots were hybridized at 42°C overnight in 5x standard saline citrate. Filters were washed twice at 65°C in 2x standard saline citrate containing 0.1% sodium dodecyl sulfate and exposed to X-ray films for 2 days at -70°C. All Northern blots were reprobed with random primed 32P-labeled human ß-actin cDNA to confirm equivalent loading of RNA samples.
MetAP2 Anti-Sense Oligonucleotides
The MetAP2 phosphorothioate anti-sense oligonucleotide (5'-AGTATTTACTTTCTCCCAAG-3') and its relative scrambled sequence (5'-CTTGGGAGAAAGTAAATACT-3') were synthesized by Sigma-Genosys Ltd. The anti-sense start position on MetAP2 mRNA coding region is 1284. This region corresponds to the large helical domain insertion on the surface of the type 2 isozyme that differentiates it from the type 1 isoform.5,6
Bcl-2 Stable Transfectants
Cells seeded at the density of 2 x 106/100-mm diameter plate were transfected by the calcium-phosphate precipitation method with a plasmid DNA containing the full-length human bcl-2 cDNA and as a selectable marker, a neomycin phosphotransferase gene. Bcl-2 expression in individually isolated clones was determined by immunoblotting with an anti-Bcl-2 antibody (N-19; Santa Cruz Biotechnology, Milan, Italy). Once a stable cell line was obtained from each clone, neomycin was removed from the culture medium. The clones were maintained in drug-free medium and Lcl-2 expression was periodically determined. As a control, cells were transfected with either an unrelated, neomycin-resistant PKR-plasmid DNA or with a plasmid DNA containing a neomycin phosphotransferase gene.
Immunoblotting and ELISA
Forty µg of protein lysates were resolved using a 10 to 12% sodium dodecyl sulfate-polyacrylamide gel and proteins transferred to polyvinylidene difluoride membranes. These were incubated for a minimum of 2 hours with either a monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology) or with a rabbit polyclonal anti-MetAP2 antibody (CM33) (Zymed Inc., Histo-Line Laboratories, Milan, Italy) and subsequently exposed to a horseradish peroxidase-conjugated secondary antibody. Bands were visualized using the ECL Plus detection kit (Amersham Corp., Milan, Italy). As an internal control the upper portion of the blot was cut and probed with an antibody recognizing an unrelated protein (ß-actin). Quantitation of Bcl-2 protein was performed using an ELISA immunoassay kit (Oncogene Science, Cambridge, MA) following the manufacture’s instructions.
Telomeric Repeat Amplification Protocol (TRAP) Assay
Either a nonamplified conventional standard or a polymerase chain reaction-ELISA-based assay (Roche Molecular Biochemicals) was used to measure telomerase activity.20,21 Cell equivalents (1 x 103 to 5 x 103) were used to visualize the DNA ladder with the standard protocol, whereas for polymerase chain reaction-ELISA, 2 x 103 cell equivalents were used. The polymerase chain reaction-ELISA protocol was provided by the assay kit manufacturer (Roche Molecular Biochemicals). Each set of TRAP assays included control reaction tubes without any extract or with RNase A (200 µg/ml)-treated extracts.
Caspase 3 Activity
Caspase 3 activity was measured using the Caspase 3 colorimetric assay kit from Clontech (Palo Alto, CA). Briefly, equal amounts of proteins from cellular lysates were added to 5 x 10-5 M substrate (Asp-Glu-Val-Asp-p-nitroanilide) in assay buffer. Samples were incubated at room temperature and analyzed using a spectrophotometer (ELISA microplate reader, Bio-Rad, Richmond, CA).
Statistical Analyses
Results are expressed as means ± SD. All statistical comparisons were made with two-sided Student’s t-test. A P value of less than 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Angiostatic agents may have a considerable impact on tumor
progression. To determine whether mesothelioma cell proliferation and
survival were affected by angiostatic molecules, we exposed
mesothelioma cells to varying concentrations of the known angiostatic
molecule, fumagillin. We observed that fumagillin potently inhibited
[3H]-thymidine uptake by mesothelioma cells,
with an apparent IC50 of 
0.1 µg/ml.
Fumagillin also significantly reduced DNA synthesis in HUVECs, but it
did not alter [3H]-thymidine uptake by normal
mesothelial cells (Figure 1A)
. To assess
selectivity of fumagillin, mesothelioma cells were exposed for
different times to a panel of well-characterized anti-angiogenic
compounds including thalidomide (10 µg/ml), angiostatin (5 µg/ml),
endostatin (10 µg/ml), and suramin (10 µg/ml). The activity of
these compounds was preliminary tested using a HUVEC proliferation
assay (results not shown). It was observed that fumagillin
time-dependently inhibited DNA synthesis in all of the mesothelioma
cell lines examined. In contrast, thalidomide, angiostatin,
endostatin, and suramin did not have any appreciable effect (Figure 1B)
. In particular, [3H]-thymidine uptake by HM
cells was reduced by 72 ± 8% after 96 hours of exposure to 0.5
µg/ml of fumagillin (P 
0.05,
fumagillin-treated versus untreated cells).
|
. Consistently,
a significant increase in nucleosome formation was detected in
fumagillin-treated HM cells (Figure 2B)
.
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. Moreover, electron
microscopy observation of ultra-thin sections of fumagillin-treated HM
cells showed a significant alteration of the mitochondrion integrity.
In particular, the presence of round osmiophilic structures, the
so-called "mitochondrial bodies," diagnostic of mitochondrial
disruption,22
was observed (Figure 3C)
.
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MetAP2 has been identified as the molecular target of
fumagillin.9,10
Therefore, we asked whether the bioactions
of fumagillin on mesothelioma cells could be related to MetAP2
expression. To this end, total RNA was extracted from malignant (MM-B1,
MM-F1, HM) and primary normal (NM-1, NM-2, NM-3) mesothelial cells and
subjected to Northern blot with specific probes for MetAP1 or MetAP2.
As shown in Figure 4A
, MM cells expressed
high MetAP2 mRNA levels, whereas lower levels were found in NM cells.
In contrast, MetAP1 was equally expressed in all cell lines. A
densitometric analysis showed that the MetAP2/ß-actin ratio was
significantly higher in malignant compared to normal mesothelial cells
(7.2 ± 1.4 versus 1.4 ± 0.62 arbitrary units;
P = 0.002) (Figure 4B)
. Consistently, MetAP2 protein
levels were significantly higher in malignant mesothelial cells
compared to normal cells (Figure 4C)
.
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To obtain a direct evidence of the relationship between MetAP2
expression and mesothelioma cell survival, a sequence-specific
phosphorothioate anti-sense oligonucleotide targeted to the coding
region of the MetAP2 gene was designed. This oligonucleotide, but not a
mismatched oligonucleotide, concentration-dependently decreased MetAP2
mRNA and protein levels in HM cells with an IC50
of 50 nmol/L (Figure 5A)
. In contrast,
it did not alter MetAP1 expression (results not shown). Maximal
inhibition was observed after 48 hours of treatment (results not
shown). When added to HM cells, the MetAP2 anti-sense oligonucleotide
time-dependently reduced cell viability with a maximum effect after 96
hours (P = 0.015, MetAP2 anti-sense
versus scrambled oligo) (Figure 5B)
, whereas it did not
alter NM-1 cell viability (result not shown). Further, the MetAP2
anti-sense oligonucleotide induced a significant nucleosome formation
in HM, but not in NM-1 cells (0.7 ± 0.052 versus
0.25 ± 0.07, optical density at 405 nm; P =
0.018) (Figure 5C)
.
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Products of genes belonging to the Lcl-2 family are main
regulators of apoptosis. In particular, Bcl-2 prevents programmed cell
death, whereas Bax can accelerate this process.23
Therefore, we asked whether a dysregulation of these genes was involved
in mesothelioma cell apoptosis after MetAP2 inhibition. To this end,
bcl-2 and bax mRNA levels were determined by
Northern blot in cells treated with either fumagillin or the MetAP2
anti-sense oligonucleotide. As displayed in Figure 6A
, fumagillin, as well as the MetAP2
anti-sense, time-dependently reduced bcl-2 expression,
whereas they did not modify bax mRNA levels in HM cells. The
scrambled oligonucleotide was ineffective (results not shown). In
contrast, MetAP2 inhibition did not determine significant changes in
either bcl-2 or bax mRNA levels in NM-1 cells. A
time-dependent inverse correlation between nucleosome formation and
Bcl-2 protein expression was also denoted in HM cells (Figure 6B)
.
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Recently, a close relationship between bcl-2 expression
and telomerase activity in cancer cells has been
denoted.24
Therefore, we hypothesized that MetAP2
inhibition might have an impact also on telomerase activity. Consistent
with previous results,25
mesothelioma cells displayed a
strong telomerase activity, whereas normal mesothelial cells did not.
When mesothelioma cells were exposed to fumagillin or the MetAP2
anti-sense a significant time-dependent reduction in telomerase
activity was observed (Figure 7A)
. A
quantitative polymerase chain reaction-ELISA-TRAP assay showed that
telomerase activity was reduced by 55% after 72 hours and by
75% after 96 hours of exposure to fumagillin
(P = 0.025 and P < 0.001,
respectively). Consistently, fumagillin-treated HM cells also exhibited
a lower number of DNA fragments, typical of telomerase activity,
compared to untreated cells (Figure 7B)
. However, telomerase mRNA
levels were not modified after exposure of HM cells to fumagillin
(results not shown).
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To further analyze the relationship between bcl-2
deregulation and reduced telomerase activity after MetAP2 inhibition,
we generated HM stable transfectants overexpressing Bcl-2. Figure 8A
shows Bcl-2 protein levels in two of
four of these clones. Clones 1 and 2 expressed threefold to fivefold
higher Bcl-2 protein levels compared to either parental HM cells or a
clone expressing an unrelated PKR/neo gene (HM-PKneo cells). Bcl-2
overexpression did not appreciably change basal telomerase activity.
However, exposure of HM-bcl-2.2 to fumagillin did not give the
inhibitory effect on telomerase activity seen with either HM or
HM-PKneo cells (Figure 8B)
. Moreover, fumagillin did not induce
apoptosis in the Bcl-2-overexpressing HM cells (Figure 8C)
.
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The cysteine proteases, caspases, are the best known effectors of
programmed cell death in most, if not all, mammalian cell
types.26
To determine whether caspases were involved in
the apoptotic pathways triggered by MetAP2 inhibition, HM cells were
exposed to the broad-spectrum irreversible caspase inhibitor,
zVAD-fmk.26
As shown in Figure 9A
, zVAD-fmk abrogated the
fumagillin-induced nucleosome formation, although it did not
prevent telomerase inhibition (results not shown). Consistently,
the MetAP2 anti-sense oligonucleotide significantly enhanced caspase
activity in HM cells, but not in the HM-bcl-2.2 clone and this effect
was blunted by zVAD-fmk (Figure 9B)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Recently, MetAP2 has been identified as the molecular
target of the angiostatic agents fumagillin and
ovalicin9,10
and it has been documented that its
inhibition by these drugs determines endothelial cell-cycle arrest in
the late G1 phase and blockage of
angiogenesis.27
For this reason, considering the relevance
of neovascularization in cancer growth and invasiveness, the fumagillin
analog, TNP-470, is being used for treatment of a variety of
neoplasms.3,4
Consistent with previous
findings,28
our present results indicate that MetAP2 may
regulate tumor growth also by a direct effect on cancer cell survival.
However, the observation that normal mesothelial cells showed very low
MetAP2 mRNA and protein levels (Figure 4)
raises the question whether
the MetAP2 gene is up-regulated as a part of the molecular events that
take place during malignant transformation of mesothelial cells. If
this is the case, MetAP2 could be proposed as a marker of mesothelial
cell malignancy and a selective pharmacological target. In this
respect, it is noteworthy that all of the mesothelioma cell lines
tested displayed very high MetAP2 mRNA levels regardless of the
histotype (Figure 3)
. However, additional studies are needed to
establish the clinical significance of MetAP2 expression in
mesothelioma. On the other hand, fumagillin inhibited DNA synthesis in
both HUVECs and mesothelioma cells (Figure 1)
, but it induced apoptosis
only in mesothelioma cells (Figure 2)
. These results suggest that the
MetAP2 signaling may involve different pathways in normal
versus malignant cells.
In mesothelioma cells MeAP2 seems to control two key regulators of
proliferation and programmed cell death, namely Bcl-2 and telomerases.
In fact, MetAP2 inhibition caused a time-dependent down-regulation of
the anti-apoptotic gene bcl-2, whereas it did not alter the
expression of the pro-apoptotic gene, bax (Figure 6)
. These
results represent the first observation of bcl-2 regulation
by MetAP2 and are consistent with the findings of membrane blebbing,
cytoplasm vacuolization, and mitochondrial body formation in
mesothelioma cells exposed to fumagillin (Figure 3)
. It is in fact
known that Bcl-2 maintains mitochondrial integrity by regulating the
opening of the transition pore, this preventing the release into the
cytosol of the caspase activators, AIF and cytochrome c.29
However, how MetAP2 regulates bcl-2 expression remains
unknown. MetAP2 is not a transcription factor, therefore, it is
unlikely that it may have a direct impact on bcl-2 gene
expression. Instead, MetAP2 controls the posttranslational processing
required for protein myristoylation.7,8
Several proteins
including members of the src tyrosine kinase family, cyclic
AMP-dependent kinase, the protein phosphatase calcineurin, and the
subunits of numerous GTP-binding proteins are modified by the covalent
attachment of myristic acid to a glycine residue, which is exposed
after removal by MetAPs of the initial amino-terminal
methionine.8
Myristoylation regulates the localization,
stability, and degradation rate of these proteins. Thus, it could be
hypothesized that in mesothelioma cells MetAP2 inhibition may alter the
function of proteins involved in the control of bcl-2
expression. Another possibility is that, because the MetAP2 inhibitor
TNP-470 induces p53 and p21
expression30
and, at least in selected systems, p53
down-regulates bcl-2 expression,31,32
the
impact of MetAP2 inhibition on bcl-2 mRNA may be in relation
with the regulation of p53. In this regard, interactions between the
p53 and Lcl-2 pathways are extremely interesting in the mesothelioma
model because it has been shown that SV40 large T-antigen, which is
likely to be involved in the pathogenesis of human mesothelioma, binds
and inactivates p53.33
Thus, in mesothelioma cells MetAP2
inhibition could restore p53 function and this may have a downstream
impact on bcl-2 expression. This hypothesis also awaits
experimental documentation.
In addition to bcl-2, MetAP2 inhibition regulated telomerase
activity in mesothelioma cells (Figure 7)
. Telomeres are structures
containing (TTAGGG)n repeats localized at the ends of chromosomes.
Their main function is to protect chromosomes from rearrangements,
degradation, and fusions. In normal somatic cells, telomere length
decreases with aging. In contrast, tumor cells express high levels of
telomerase, a ribonucleic acid-protein complex that prevents telomere
loss. Moreover, telomerase inhibition may alter cancer cell
growth.24
Recently, telomerase activity was detected in
>90% of mesothelioma, but not in normal mesothelial cells, indicating
that telomerase re-activation may occur during the development of this
tumor.25
This observation, together with our present
results showing that mesothelioma cells express high MetAP2 mRNA and
protein levels (Figure 4)
and that in these cells MetAP2 inhibition
significantly reduced telomerase activity (Figure 7)
, seems to indicate
that a close relationship may exist between MetAP2 expression and
telomerase re-activation in human mesothelioma. Whether or not this is
a general phenomenon during carcinogenesis remains to be determined.
However, this previously unappreciated MetAP2 effect on telomerase
activity may be relevant to selectively direct the pharmacological
effects of MetAP2 inhibitors toward actively replicating cells, such as
cancer cells, that express high telomerase activity.
It has been reported that telomerase activity is increased by
bcl-2 overexpression in human cancer cells with low
expression of this gene.24
Moreover, bcl-2
overexpression in pheochromocytoma cells protects them from the
apoptosis induced by telomerase inhibitors.34
We observed
that bcl-2 overexpression in mesothelioma cells did not
change their already quite high basal telomerase activity, however, it
did counteract the effects of MetAP2 inhibition on telomerase activity
and apoptosis (Figure 8)
. Taken together, these results indicate that
in mesothelioma cells Lcl-2 may function as a downstream effector of
MetAP2 actions on apoptosis and telomerase activity. On the other hand,
members of the caspase family are in many circumstances ultimately
responsible for DNA fragmentation and apoptosis.31,32
Because the caspase inhibitor zVAD-fmk abrogated mesothelioma cell
apoptosis induced by MetAP2 inhibition and a MetAP2 anti-sense
oligonucleotide increased caspase activity (Figure 9)
, it may be
concluded that caspases are involved in MetAP2 regulation of
mesothelioma cell survival. However, the MetAP2 anti-sense did not
modify caspase activity in Bcl-2 overexpressing tumor clones (Figure 9)
and zVAD-fmk did not reverse the fumagillin-dependent inhibition of
telomerase activity (results not shown). Taken together these findings
indicate that MetAP2 acts upstream of Bcl-2, whereas Bcl-2 site of
action is likely to be upstream of that of telomerases and caspases. On
the other hand, telomerases seem to act upstream of caspases. A
schematic representation of this pathway is shown in Figure 10
.
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Footnotes |
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Supported by grants from Associazione Italiana Ricerca contro il Cancro and Ministero dell’Universtà e Ricerca Scientifica, ex 60%, ex 40% (to A. P.).
Accepted for publication April 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/colchicine combined treatment on Burkitt lymphoma cells: molecular and ultrastructural changes. Cytokine 1999, 2:144-150
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). Scale
bar, 0.5 µm.
