escalating-dose prednisolone treatment on an acute

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SUPPLEMENTARY DATA TO THE ARTICLE TITLED:
“PREDNISOLONE EXERTS LATE MITOGENIC AND BIPHASIC EFFECTS ON RESISTANT
ACUTE LYMPHOBLASTIC LEUKEMIA CELLS: RELATION TO EARLY GENE EXPRESSION”
Running Title: Prednisolone early effects on an acute lymphoblastic leukemia cell line.
George I Lambrou1, Spiros Vlahopoulos1, Chrisanthi Papathanasiou1, Maria Papanikolaou1, Michael
Karpusas2, Emmanouil Zoumakis3, Fotini Tzortzatou-Stathopoulou1
1
Hematology/Oncology Unit, First Department of Pediatrics, University of Athens Medical School,
“Aghia Sophia” Children’s Hospital, Athens, Greece
2
University Research Institute for the Study and Treatment of Childhood Genetic and Malignant Diseases
3
Choremeio Research Laboratory, First Department of Pediatrics, University of Athens Medical School,
“Aghia Sophia” Children’s Hospital, Athens, Greece
DIFFERENTIALY EXPRESSED GENES DESCRIPTION
Differentially expressed genes
GCs are known to lead to chromatin modification about two hours following treatment.[1] A detectable
change in the expression of genes that are immediately regulated by the GC is therefore expected to reach
the stage of mRNA steady-state levels at approximately three to four hours after treatment. Then, genes
regulated by the GC, begin to influence subsequent gene expression. cDNA microarray analysis upon 4 h
of treatment has been chosen, in order to reduce the complexities that arise later due to ensuing feedback
mechanisms, at the same time focusing on the GR/NF-κB-linked, direct target genes.
The 10nM and 700μM prednisolone concentrations were chosen because they correspond clearly to the
extreme effects, i.e. anti-apoptosis accompanied with mitogenic effect and cytotoxicity accompanied with
resistance, as observed by flow cytometry. Moreover, the high concentration (700μM) used, is similar to
concentrations used in different studies in CCRF-CEM cells [2] as well as primary cell cultures derived
from childhood ALL patients[3].
This analysis was expected to reveal key genetic regulators of further downstream pathways.
Analysis of the microarray data identified 40 genes that were differentially expressed and were sorted by
function as follows: apoptosis-related genes, tumor-suppressor genes, cell cycle and proliferation-related
genes, cancer preventing genes, metabolism-related genes, intra- and extra-cellular signaling molecules
and genes whose function could not be interpreted in the context of the present model. Chromosome maps
of the differentially expressed genes, predicted that prednisolone affects genes in almost all chromosomes
(Figure 6A). Here, the differentially expressed genes are discussed in the context of the system of study.
1
The effects of the low prednisolone dose
The low dose of prednisolone appeared to regulate the following genes: BIRC3 (Accession
AF070674) and CDC25C (Accession NM_001790) were downregulated by prednisolone, while GEM
(Accession NM_005261), GUK1 (Accession NM_000858), PCTK1 (Accession NM_033018), OSTF1
(Accession NM_012383), IGF1 (Accession X57025), MMP19 (Accession NM_022790), TGFA (Accession
NM_003236), DLEU1 (Accession NM_005887), CDC6 (Accession NM_001254) were upregulated.
Apoptosis-Related/Tumor Suppressor genes: GRB10 (Accession D86962) and BIRC3 belong to
cluster 7 indicating a common regulatory mechanism by prednisolone. GRB10, shows a biphasic
expression profile. It is marginally downregulated by the low dose but as it will be discussed further on it
is upregulated by the high prednisolone dose. It is involved in apoptosis-evasion since its deficiency
sensitizes cells to apoptosis.[4] BIRC3 is a target of NF-κB (RelA) which inhibits apoptosis.[5] BIRC3
expression, has been reported to be induced in a sensitive CCRF-CEM subclone[6] but suppressed in the
present resistant cell line. BIRC3 is a caspase inhibitor; its downregulation by prednisolone hints to the
apoptosis-inducing mechanism of glucocorticoids on leukemic cells, but it is evidently not sufficient since
cells exhibit resistance. Similarly, GRB10 does seem to participate to the observed resistance as it is
upregulated by the high prednisolone dose. This indicates the existence of a dual mechanism in the control
of apoptotic pathways. PRAME (Accession NM_006115), DAP (Accession NM_004394), GLG1
(Accession NM_012201), TIE1 (Accession NM_005424), all apoptosis-related genes, remain unaffected by
the low dose of prednisolone. DLEU1, a tumor-suppressor gene, seems to be upregulated by the low-dose
of prednisolone and remain unaffected by the high dose. This explains partly the exhibited resistance at
the high dose but not the apoptosis-evasion observed at the low dose.
Cell Cycle genes: CDC25C, CDC25B (Accession NM_021874) (significantly expressed, but not
consistent along the three experiments) and CDC6 are cell cycle related genes regulated by the low
prednisolone dose. CDC25C and CDC25B are downregulated which is consistent with the expected effect
of prednisolone but not to the observed i.e. the mitogenic effect induced by the low doses. On the other
hand, they are evenly regulated by the high dose of prednisolone which is consistent with the proliferative
effect of the high doses compared to untreated cells. Specially, CDC25B is a steroid receptor coactivator
that mediates interactions with CREB binding protein (CBP) [7, 8] which is a GR regulator.[9] The fact
that CBP is present in limiting amounts in a cell, thereby subject to squelching, when levels of a partner
protein exceed a certain value, makes CDC25B a potential gene target that would be consistent with a dual
effect of prednisolone. The effect of low concentration is balanced when high dose of GC is used:
CDC25B may exert opposite effects when CBP levels are squelched by high levels of activated GR. On
the other hand, high levels of activated GR may also repress CDC25B gene expression by binding and
inactivating transcription factor NF-κB RelA. Also, CDC25C plays an important role in the transition to
G2/M phase.[10, 11] Its under-expression by low dose of prednisolone does not explain cell cycle
progression while high dose of prednisolone leaves CDC25C levels unaffected. It has been reported
though that downregulation of CDC25C is related to G2/M arrest in a different system.[12] CDC6, along
2
with P1-Cdc21 (Accession X74794) (MCM4), which belongs to the family of MCMs, are participating in
DNA replication procedures.[13] Prednisolone upregulates CDC6 (Accession NM_001254) at both
concentrations while leaves unaffected the P1-Cdc21 (Accession X74794) at low dose and marginally upregulates it at high prednisolone dose. This result explains the shift to S-phase 24h after prednisolone
exposure at all concentrations except the higher ones. Further on, it has been reported that v-Myb and
CDC6 (Accession NM_001254) are regulated by E2F.[14] The levels of these genes are critical for the
transition to the S-phase and DNA synthesis, justifying an early and rapid GR regulation.
Metabolism-related genes: The appearance of the metabolism-related genes PCTK and GUK1
may provide compelling reasons for the enhanced proliferative behavior induced by the low prednisolone
doses. Also, the involvement of metabolism-related genes supports the hypothesis presented earlier,
possibly explaining the necrotic effects at low doses after 72 h of treatment. The selective expression of
certain metabolic genes supports the demands for increased growth, but on the other hand poses a
challenge for the capability of cell physiology to respond to the increased proliferation in a coordinated
manner. In a portion of the cells, this metabolic tension apparently leads to necrotic death, due to lack of
capability to integrate and absorb all changes in regulatory, structure-maintaining and energy-generating
reaction pathways. The upregulation of these metabolism-related genes supports the observation of
increased proliferation, setting increased metabolic demands to the cell machinery. PCTK1 is up-regulated
by the low prednisolone dose while remains unaffected by the high dose. GUK1 is upregulated by both
prednisolone doses. GUK1 is reported to participate in cytotoxicity regulation to cis-platin in
prokaryotes.[15] There are no reports concerning its role in leukemia or GR-regulation. Especially, the
G6PD (Accession NM_000402) gene which was significantly expressed (p<<0.0 0001) but did not pass
the test for consistency among the three experiments, is reported to be regulated by the transcription factor
CRE-BP1 (ATF2) and it is regulated in a bi-phasic manner through two distinct genetic elements.[16]
Extra- and Intra-cellular Signaling genes: TGFA, IGF1 and GEM are extra- and intracellular
signaling molecules. TGFA, is an extracellular factor which competes with EGF for EGFR. It is known to
stimulate differentiation and mitogenesis. It is upregulated in the present system in both prednisolone
doses compared to untreated cells, which justifies the mitogenic effect induced, especially, by the low
prednisolone dose. Up to date there are no reports concerning the role of GCs or GR in the regulation of
this gene. Interestingly, it has been reported that TGFA, when expressed, prevents glucocorticoid-induced
suppression in a rat cell system.[17] TGFA upregulation in the present system hints to its possible role in
apoptosis-evasion. IGF1, is an extracellular signal upregulated by prednisolone in the present system. It
has been reported to interfere with CDC25C, GRB10, MAP3K5[18] and TIE1 in breast cancer, regulating
cell proliferation and apoptosis.[19, 20] Also, it has been reported that Myb regulates pathways
participating in IGF1 expression.[21] Dexamethasone upregulates the mRNA levels of IGF1, as reported
previously.[22] Interestingly, IGF1 is suppressed by inflammation, an effect which is reversed by
glucocorticoid treatment[23] and antagonizes with dexamethasone for muscular growth pathways.[24] In
the present resistant system its upregulation suggests a role in cell’s growth. GEM belongs to the
3
RAD/GEM family of GTP-binding proteins. It is associated with the inner face of the plasma membrane
and could play a role as a regulatory protein in receptor-mediated signal transduction. It has been reported
that it is expressed in NK cells and some T-cells.[25] There are no known relations of GEM to leukemia or
the synergism with glucocorticoids.
OSTF1, Osteoclast-stimulating factor-1 is an intracellular protein produced by osteoclasts that
indirectly induces osteoclast formation and bone resorption.[26] There are no known relations of OSTF1
to leukemia, glucocorticoids or the present cell system, which makes this gene an interesting factor for
future studies. Additionally, MMP19 belongs to the matrix metalloproteinase (MMP) family, involved in
the breakdown of extracellular matrix in processes, such as arthritis and metastasis. Most MMP's are
secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. This
protein is expressed in human epidermis and it has a role in cellular proliferation as well as migration and
adhesion to type I collagen. It has been reported that MMP19 is a ‘late’, protein synthesis-dependent
gene[27] and it is required for proper T-cell development and growth.[28] Up-to-date no relations are
known to glucocorticoids or leukemia.
Summarizing, the microarray analysis showed the TGFA, IGF1, GEM and MMP19 as possible
candidate genes for the exhibited mitogenic effect of the low prednisolone dose. Also, the fact that BIRC3
was down-regulated in this resistant cell line while it is up-regulated in the sensitive CCRF-CEM clone, as
previously reported[6], consists it also as a possible candidate for early determination of resistance to
glucocorticoids.
The effects of the high prednisolone dose
The high prednisolone dose regulated the following genes: CDC42BPA (Accession NM_014826), EPHB6
(Accession NM_004445), NOMO1 (Accession NM_014287), PHB2 (Accession NM_007273) are downregulated by the high prednisolone dose while TGFA (Accession .NM_003236), GRIM19 (Accession
NM_015965), NHS (Accesion NM_198270), GRB10 (Accession D86962), GUK1 (Accession
NM_000858), NPAL2 (Accession NM_024759), MMP19 (Accession NM_022790), ), MAP3K5 (Accession
NM_005923), CCL25 (Accession NM_005624), YWHAZ (Accession NM_003406), IGF1
(Accession
X57025), GEM (Accession NM_005261), FBX14 (Accession NM_024735), LFNG (Accession BC014851),
PTPRF (Accession NM_002840), VIL2 (Accession NM_003379), ALOX5 (Accession NM_000698), CDC6
(Accession NM_001254), TIE1 (Accesion NM_005424) are up-regulated by the high prednisolone dose.
Apoptosis-Related/Tumor Suppressor genes: GRB10, is involved in apoptosis-evasion since its
deficiency sensitizes cells to apoptosis.[4] Therefore, its overexpression hints to a possible candidate for
the exhibited resistance to prednisolone. GRIM19 is considered to be a cell death regulatory protein[29]
and a factor required for NF-κB activation.[30-32] However, upregulation by the high dose of
prednisolone does not explain the resistant effect. It was also predicted that the GRIM19 gene possesses a
binding site for CREB transcription factor (Table 2). There are no reports concerning this prediction, it is
however a hint towards the fact that GRIM19 is a target of GR. This was also confirmed by the qRT-PCR
4
at 4h (Figure 9A). However, the surprise came from the 48h test were GRIM19 expression reversed
(Figure 8B) to fit the observed results in cell death at 72h (Figure 2D). GRIM19 has not been previously
reported to be associated to GCs or GR. PTPRF, up-regulated by the high prednisolone dose, encodes a
protein which is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be
signaling molecules that regulate a variety of cellular processes including cell growth, differentiation,
mitotic cycle, and oncogenic transformation. This PTP possesses an extracellular region, a single
transmembrane region, and two tandem intracytoplasmic catalytic domains, and thus represents a
receptor-type PTP. This PTP was shown to function in the regulation of epithelial cell-cell contacts at
adherents junctions, as well as in the control of beta-catenin signaling. It is a candidate tumor suppressor
gene manifesting similar behavior to GRIM19 that is, unaffected by the low prednisolone dose and upregulated by the higher dose. Interestingly, it was predicted by TFBM analysis that these two genes do not
share a common regulatory but are still commonly regulated as it appears from clustering analysis (Figure
5). Finally, TIE1, a membrane receptor reported to play a role in leukemic cell survival.[33] Prednisolone
exerts a dual effect; the low dose does not affect TIE1 and the high dose upregulates it. Again, the upregulation of TIE1 in concordance with the fact that this is a membrane receptor makes it a candidate
molecule for the observed resistance and also makes it a possible marker for early resistance detection.
There are no reports to the present suggesting a connection between TIE1 and resistance to
glucocorticoids. Along with TIE1, EPHB6 is a membrane receptor found to be overexpressed in AML[34]
and its overexpression is linked to good prognosis in neuroblastoma.[35] EPHB6, is however downregulated by the high prednisolone dose suggesting a possible role in glucocorticoid resistance. These
findings suggest that prednisolone works in a dose-dependent dual mode at the early response activating
different GR transactivation or transrepression pathways. This dual mode is reflected on the observed
duality of prednisolone late response at 72 h of treatment.
Cell Cycle genes: Not much is known about the CDC42BPA gene and there are no reports
concerning its regulation by glucocorticoids. It has been reported though that the cdc42 kinase plays an
important role in cell polarity, motility.[36] In the present system it seems that CDC42BPA is regulated by
prednisolone as it decreases its mRNA levels compared to control consistently along the three
experiments. Prednisolone up-regulates CDC6 at both concentrations while leaves unaffected the P1Cdc21 at low dose but up-regulates it at high prednisolone dose, as discussed previously. PHB2 is a
mitochondrial located complex which controls cell proliferation and cell compartmentalization. It is
reported that its loss (i.e. down-regulation) is connected to impaired cell proliferation and apoptosis
evasion.[37, 38] Consistent to the observed apoptosis and cell proliferation in the present system since it is
downregulated by the high prednisolone dose and remains unaffected by the low dose.
Metabolism-related genes: The upregulation of these metabolism-related genes supports the
observation of increased proliferation, setting increased metabolic demands to the cell machinery. GUK1
and ALOX5 are upregulated by prednisolone while ATP5F1 (Accession NM_001688) remains unaffected
by both prednisolone concentrations and PCTK1 is up-regulated by the low prednisolone dose.
5
Extra- and Intra-cellular Signaling genes: TGFA and IGF1 are interestingly up-regulated by the
high prednisolone dose as it is by the low dose. This suggests that they play a role in glucocorticoidinduced apoptosis evasion. Interestingly, low dose prednisolone leaves CCL25 unaffected while it is upregulated by the high dose, probably contributing to the observed resistance. The cytokine encoded by this
gene displays chemotactic activity for dendritic cells, thymocytes, and activated macrophages but is
inactive on peripheral blood lymphocytes and neutrophils. The product of this gene binds to chemokine
receptor CCR9. CCL25 is reported to rescue T-cell ALL cells from TNF-α mediated apoptosis[39]
suggesting a similar function with glucocorticoids. An interesting gene is VIL2 (ezrin). The protein
encoded by this gene is a connector between cytoplasmic receptors such as CD43, CD44 and the
cytoskeleton. This protein has been reported to be expressed in the CCRF-CEM cell line and overexpressed in the Raji cell line. High levels of ezrin are associated with metastatic and proliferative
behavior.[40] This is consistent with the results of the present study indicating a novel role of VIL2 in the
proliferation and survival of glucocorticoid-treated leukemic cells. GEM is also up-regulated by the high
prednisolone dose as reported for the low prednisolone dose. MAP3K5 is part of the MAPK pathway. It
does not activate MAPK/ERK pathway but instead it activates the JNK kinase. There are numerous
reports about the relationship of MAP3K5 and JUN kinase as well as c-jun. Interestingly, in the present
system it seems that prednisolone exhibits a biphasic effect on the MAPK pathway. The low dose
marginally downregulates the MAP3K5 gene while the high dose upregulates this gene. This hints to the
late biphasic effect
of prednisolone since the JNK pathway, activated by MAP3K5 is involved in
apoptosis control.[18] Also, as it has been reported, it interferes with IGF1, GRB10, CDC25C and TIE1 to
regulate apoptosis and cell cycle. All these genes are up-regulated by the high prednisolone dose except
CDC25C suggesting a survival-induced mechanism along with G2/M arrest due to CDC25C downregulation. YWHAZ, gene belongs to the 14-3-3 family of proteins which mediate signal transduction by
binding to phosphoserine-containing proteins. It regulates insulin sensitivity which relates it to the
IGF1factor revealed by the microarrays.[41] Gene ontology analysis has shown relation of YWHAZ to
VIL2, c-jun, CREB1, TIE1, ALOX5, GR, RelA, ATF2, TGFA, MYB and CDC25C (see gene ontology
link).
No connection between NOMO1, NPAL2, NHS, LFNG and glucocorticoids or leukemia has
been reported. NOMO1 encodes a protein originally thought to be related to the collagenase gene family.
This gene is one of three highly similar genes in a region of duplication located on the p arm of
chromosome 16. These three genes encode closely related proteins that may have the same function. The
protein encoded by one of these genes has been identified as part of a protein complex that participates in
the Nodal signaling pathway during vertebrate development. NHS (Nance-Horan syndrome/congenital
cataracts and dental anomalies) encodes a protein containing four conserved nuclear localization signals.
The encoded protein may function during the development of the eyes, teeth, and brain. Mutations in this
gene have been shown to cause Nance-Horan syndrome. LFNG is a gene encoding a member of the
glycosyltransferase superfamily. The encoded protein is a single-pass type II Golgi membrane protein that
6
functions as a fucose-specific glycosyltransferase, adding an N-acetylglucosamine to the fucose residue of
a group of signaling receptors involved in regulating cell fate decisions during development. However,
these four genes are involved in developmental regulation suggesting that glucocorticoids regulate also
genes participating in early development. The FBX14 gene is a member of the F-box protein family,
characterized by an approximately 40-amino acid F-box motif. SCF complexes, formed by SKP1 (MIM
601434), cullin and F-box proteins, act as protein-ubiquitin ligases. F-box proteins interact with SKP1
through the F box, and they interact with ubiquitination targets through other protein interaction
domains[42] also reported to be a potent tumor suppressor gene.[43] Finally, MMP19 remains upregulated and by the high prednisolone dose as by the low one reinforcing its role in glucocorticoid
regulation.
Summarizing, the microarray results showed that the GRB10, TIE1, EPHB6, PHB2, TGFA,
CCL25, IGF1 and VIL2 (ezrin), might consist of possible early candidates for the detection of
glucocorticoid resistance and apoptosis evasion.
7
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