camp dependent protein kinase (pka)-mediated c-myc...

MOL # 97915

1

cAMP dependent Protein kinase (PKA)-mediated c-Myc degradation is dependent on the relative proportion of PKA-I and PKA-II isozymes. Qingyuan Liu, Eric Nguyen, Stein Døskeland, Évelyne Ségal-Bendirdjian

INSERM UMR-S 1007, Homéostasie Cellulaire et Cancer, Université Paris-Descartes, Paris

Sorbonne Cité, Paris, France (Q. L., E. N., E. S-B.)

Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009, Bergen,

Norway (S. D).

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on June 23, 2015 as DOI: 10.1124/mol.115.097915

at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from

http://molpharm.aspetjournals.org/

MOL # 97915

2

Running title: PKA-I/PKA-II ratio determines c-Myc stability.

Corresponding author: Évelyne Ségal-Bendirdjian, INSERM UMR-S 1007, Université Paris-

Descartes, 45 rue des Saints-Pères, 75006 Paris, France.

Tel: 33 1 42 86 22 46; Fax: 33 1 42 86 21 62; Email: [email protected]

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Text Pages: 28

Tables: 0

Figures: 5

References: 63

Supplemental Figures : 1

Words in Abstract: 182

Introduction: 545

Discussion: 788

Abbreviations:

AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; PKA, cAMP-dependent

protein kinase A; PKA-RI, regulatory subunit I of PKA; PKA-RIIα and -RIIβ, regulatory subunit

α and β of PKA; PKA-I/II, Protein kinase type I/II; APL, acute promyelocytic leukemia; ATRA,

all-trans retinoic acid; RARα, retinoic acid receptor-α; 8-CPT-cAMP, 8-(4-chlorophenylthio)

adenosine 3’,5’-cyclic mono phosphate; 2-Cl-8-AHA-cAMP, 2-chloro-8-aminohexylamino-

cAMP; N6-Bz-8-Pip-cAMP, N6-benzoyl-8-piperidino-cAMP; Rp-8-Br-cAMPS, 8-

bromoadenosine-3’,5’-cyclic monophosphorothioate, Rp-isomer; NBT, p-Nitro-Blue

tetrazolium; PMA, Phorbol 12-myristate 13-acetate, 10058-F4 : [2,E]-5-[4-ethylbenzylidine]-2-

thioxothiazolidin-4-one.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

3

ABSTRACT

The transcription factor c-Myc regulates numerous target genes that are important for

multiple cellular processes such as cell growth and differentiation. It is commonly

deregulated in leukemia. Acute promyelocytic leukemia (APL) is characterized by a blockade

of granulocytic differentiation at the promyelocyte stage. Despite the great success of ATRA-

based therapy, which results in a clinical remission by inducing promyelocyte maturation, a

significant number of patients relapse due to the development of ATRA-resistance. A

significant role has been ascribed to the cAMP-PKA signaling pathway in retinoid treatment

since PKA activation is able to restore differentiation in some ATRA-resistant cells and also

eradicate leukemia initiating cells in vivo. In this study, using NB4 APL cell variants resistant

to ATRA-induced differentiation, we reveal distinct functional roles of the two PKA isozymes,

PKA-I and PKA-II, on the steady-state level of c-Myc protein, providing a likely mechanism by

which cAMP elevating agents can restore differentiation in ATRA-maturation resistant APL

cells. Therefore both the inhibition of c-Myc activity and the PKA-I/PKA-II ratio should be

taken into account if cAMP-based therapy is considered in the clinical management of APL.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

4

INTRODUCTION

The basic helix-loop-helix leucine zipper transcription factor c-Myc regulates the expression

of multiple target genes involved in cell cycle progression, metabolism, differentiation,

proliferation, and apoptosis (Bernard and Eilers, 2006; Dang, 1999; Dang et al., 2009; Dang

et al., 2006; Hoffman and Liebermann, 2008; Jiang et al., 2008; Schmidt, 1999). c-Myc

increases the expression of the hTERT gene coding for the catalytic subunit of telomerase

(Oh et al., 1999; Wu et al., 1999), whose activity is required for the unlimited growth potential

of tumor cells (Greider, 1999) while it downregulates the C/EBPβ transcription factor that

promotes the differentiation of numerous cell types, including the final maturation of acute

promyelocytic leukemia (APL) cells (Duprez et al., 2003; Mink et al., 1996) (Pan et al.,

2014). Because of its role in multiple cellular pathways, a tight control of c-Myc level is

required. This is achieved through multiple mechanisms acting at transcriptional, post-

transcriptional, translational, and post-translational levels (Dani et al., 1984; Lemm and Ross,

2002; Malempati et al., 2006; Sears, 2004; Wall et al., 2008). Recently, c-Myc was reported

to be subjected to complex regulation at both the transcriptional and post-translational levels

by cAMP-dependent protein kinase (PKA), in a PKA catalytic subunit isoform (PKA-Cα, PKA-

Cβ) specific manner (Padmanabhan et al., 2013).

PKA is the major intracellular target of cAMP. At low cAMP concentration, PKA exists mainly

as an inactive tetrameric holoenzyme composed of two regulatory (R) and two catalytic (C)

subunits. Activation of PKA occurs when four molecules of cAMP bind to the R subunits,

promoting the dissociation of the PKA holoenzyme into two active catalytic subunits and a

dimer of regulatory subunits (Taylor et al., 2013). The two PKA isozymes differ in their

regulatory subunits (R subunits): PKA type I (PKA-I) containing RIα or RIβ and PKA type II

(PKA-II) containing RIIα or RIIβ. The importance of the relative cellular ratio between PKA-RI

and PKA-RII has already been reported supporting the idea that specific functions can be

assigned to PKA isozymes mediating the distinct effects of cAMP in cellular processes such

as growth and differentiation (Cho-Chung et al., 1995; Ji et al., 2008; Kopperud et al., 2003;


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

5

Ogreid et al., 1989; Pidoux and Tasken, 2010; Rohlff et al., 1993; Schwartz and Rubin,

1985).

Cyclic AMP signalling has a key role in retinoid treatment efficacy in APL. Indeed, APL

differentiation induced by all-trans retinoic acid (ATRA) can be enhanced by activators of

cAMP signaling. Furthermore, differentiation can be restored in some ATRA-resistant APL

cells if ATRA is combined with cAMP analogs (Guillemin et al., 2002; Kamashev et al., 2004;

Quenech'Du et al., 1998; Ruchaud et al., 1994).

Using the NB4 cell line, a model of acute promyelocytic leukemia (APL), and its variant NB4-

LR1 subline, which is resistant to ATRA-induced maturation, we found previously that NB4-

LR1 cells have a shift in isozyme composition expressing more PKA-I and less PKA-II than

NB4 cells. In addition, using isozyme-selective pairs of cAMP analogs, we showed that

activation of both PKA-I and PKA-II is required for optimal stimulation of ATRA-induced

differentiation (Nguyen et al., 2013). The present study uses the above cell lines that differ in

their PKA-I/PKA-II ratio to reveal the respective role of PKA-I and PKA-II for c-Myc protein

stability and the relation between c-Myc expression, the expression of C/EBPβ and ATRA-

induced maturation.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

6

MATERIALS AND METHODS

Reagents. 8-(4-Chlorophenylthio)adenosine 3’,5’-cyclic adenosine mono phosphate (8-CPT-

cAMP), all trans retinoic acid (ATRA), p-Nitro-Blue tetrazolium chloride (NBT), Phorbol 12-

myristate 13-acetate (PMA), 10058-F4 ([2,E]-5-[4-ethylbenzylidine]-2-thioxothiazolidin-4-one)

and benzoyloxycarbonyl-Leu-Leu-Leu-aldehyde (MG132) were from Sigma (St. Louis, MO).,

N6-Mono-tert-butylcarbamoyl-cAMP (N6-MBC-cAMP), 2-Chloro-8-Aminohexylamino-cAMP

(2-Cl-8-AHA-cAMP), N6-benzoyl-8-piperidino-cAMP (N6-Bz-8-Pip-cAMP), and Rp-8-

Bromoadenosine 3’,5’-cyclic monophosphate (Rp-8-Br-cAMPS) were from Biolog (Bremen,

Germany).

Cell Lines and Cell Culture. NB4, NB4-LR2 and NB4-LR1SFD cell lines were cultured as

previously reported (Duprez et al., 2000; Lanotte et al., 1991; Pendino et al., 2002) in RPMI

1640 medium supplemented with 10 % foetal bovine serum (PAA Laboratories, Pasching,

Austria), penicillin (100 IU/ml), streptomycin (100 mg/ml), L-glutamine (2 mM) and sodium

bicarbonate (0.75 mg/ml), and incubated at 37°C in a 5% CO2 atmosphere.

Viral knockdown of PKA-RIIα and PKA-RIIβ. The establishment of PKA-RIIα, PKA-RIIβ,

and PKA-RIIαβ double knockdown cells was previously described (Gausdal et al., 2013). For

PKA-RIIα knockdown, the lentiviral plasmid pLKO.1/shRNA/PKAR2A, CSHGLYC-

TRCN0000037806 was provided by Sigma. For knockdown of PKA-RIIβ, the PRKA-RIIβ

targeting sequence (hairpin loop in small letters) 5’-

GGCCTTAATGTACAATACACCAGAGCAGgaccaggaccagCTGCTCTGGGTGTATTGTACAT

TAAGGCC-3’ was inserted in the retroviral vector RRI-Green/L071 (provided by Dr. D.

Micklem and Dr. J. Lorens, University of Bergen, Norway) downstream of a modified human

U6 promoter. Control vectors (pLKO.1/TRC and pLKO.1/shRNA/scramble controls) were

kindly provided by David Root (Moffat et al., 2006) and David M. Sabatini (Sarbassov et al.,


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

7

2005) (both from Massachusetts Institute of Technology, Cambridge, MA) through a material

transfer agreement (Addgene plasmids10879 and 1864).

Whole Cell Extracts and Western Blot Analysis. Cells were washed twice with ice cold

PBS and lysed in 8 % SDS, 0.25 M Tris pH 6.8, 1% protease inhibitor cocktail (Sigma,

#P8340), 2.5 mM NaF and 0.1 mM sodium orthovanadate. The lysates were vortexed and

boiled twice for 5 min. Insoluble material was removed by centrifugation at 18,000 g for 1 h.

Protein concentration was determined using BCA protein assay (Thermo Scientific, Rockford,

IL). Total cellular proteins were separated by SDS-polyacrylamide gel electrophoresis

according to Laemmli (Laemmli, 1970). Total cellular proteins separated by SDS-

polyacrylamide gel electrophoresis (PAGE) were transferred onto polyvinylidene fluoride

(PVDF) membranes. Blots were blocked with 5% non-fat dry milk, 0.5% Tween 20 in PBS pH

7.4 (PBS-Tween-milk), and then probed with one of the following primary antibodies: anti-

PKA-RIα, anti-PKA-RIIβ, anti-PKA-C, anti-c-Myc (BD Biosciences, Franklin Lakes, NJ,

#610609, #610625, #610980, #554207, respectively), anti-PKA-RIIα (Santa Cruz

Biotechnology, Santa Cruz, CA, sc908), anti-Lamin B (Santa Cruz Biotechnology, Santa

Cruz, CA, sc-6217), anti-CEBPβ (Abcam, 32358), and CD11c (52632, Abcam) diluted in

PBS-Tween-milk. After washing, blots were incubated with the appropriate peroxidase-

conjugated secondary antibody diluted in PBS-Tween-milk and washed again. Peroxidase

activity was detected by chemiluminescence (Western Lighting Plus-ECL, Perkin Elmer,

Waltham, MA) and pictures were taken with a cooled CCD camera (Image Quant LAS 4000,

GE Healthcare). The bands were quantified with Image Quant TL software.

RNA Preparation and Quantitative Reverse-Transcription Polymerase Chain Reaction.

Total cellular RNA was collected from samples using TRIzol reagent (Life Technologies,

Carlsbad, CA) as described by the manufacturer. Reverse transcription using Transcriptor


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

8

First Strand cDNA kit (Roche Diagnostics, Basel, Switzerland) and quantitative real-time

PCR using the LightCycler technology and the LightCycler FastStart DNA MasterPLUS

SYBR Green kit were performed as described before (Deville et al., 2011). c-Myc specific

primers sequences were 5’-GCTCTCCTCGACGGAGTCC-3’ for the sense primer located in

exon 3 and 5’- CCACAGAAACAACATCGATTTCTT-3’ for the antisense primer located in

exon 4. c-Myc mRNA level was normalized to the expression of GAPDH mRNA measured in

parallel using a sense primer 5’-CACCCATGGCAAATTCCATGGC-3’ and an antisense

primer 5’-GCATTGCTGATGATCTTGAGGCT-3’ located in exon 6 and exon 8, respectively.

Cell maturation assays. NB4 cells committed into the granulocytic differentiation pathway

can be identified before morphological changes by probing their ability to undergo an

oxidative burst after activation with the phorbol 12-myristate 13-acetate (PMA). This oxidative

burst was evaluated by microscopy after performing the NBT reduction assay (Pick, 1986).

Statistical analysis. Data are presented as mean ± SEM of at least three independent

experiments. Statistical analyses were performed using GraphPad Prism 5.0 (two-tailed

unpaired student’s t-test where * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

9

RESULTS

8-CPT-cAMP induces the proteasomal degradation of c-Myc in the ATRA-maturation

resistant NB4-LR1SFD cells.

In our effort to characterize the mechanisms responsible for the resistance of the NB4-LR1

cells to ATRA-induced differentiation and its restoration upon activation of cAMP signalling,

we have recently shown an altered balance between PKA-I and PKA-II in these resistant

cells compared to the parental NB4 cells (Nguyen et al., 2013). In fact, NB4-LR1 cells have

decreased expression of both PKA-RIIα and PKA-RIIβ isoforms. This finding and the recent

observation that PKA activity influences the steady-state levels of c-Myc (Padmanabhan et

al., 2013) prompted us to determine the level of c-Myc expression in the NB4 cell model

upon activation of cAMP signalling. We use here the NB4-LR1SFD cell line, which is derived

from the NB4-LR1 cell line upon continuous exposure to ATRA (Pendino et al., 2002). This

cell line retains all the features of the NB4-LR1 cell line in terms of resistance to ATRA-

induced maturation and its restoration upon activation of PKA. It differs, however, from the

NB4-LR1 cell line in being resistant to telomerase dependent cell death induced by long-term

ATRA treatment This cell variant is therefore a suitable cellular model to study the

mechanisms of resistance of APL to ATRA-based therapies. Figure 1 shows that, as already

reported for the maturation-resistant NB4-LR1 cells (Nguyen et al., 2013), the NB4-LR1SFD

cells have a PKA isozyme switch compared to the NB4: they have a decreased content of

both PKA regulatory subunits IIα and IIβ while PKA regulatory subunit I shows a

compensatory increase. In contrast, the ATRA-maturation resistant cell line NB4-LR2, which

owes its resistance to a PML-RARα mutation without impaired of cAMP signalling (Duprez et

al., 2000), had unaltered PKA isozyme distribution: NB4 and NB4-LR2 cells expressed both

the RIIα and RIIβ subunits of PKA-II and low levels of the RI regulatory subunit of PKA-I.

Therefore, the constitutive PKA-I/PKA-II ratio is much higher (> two order of magnitude;

Figure 1B) in NB4-LR1SFD cells than in NB4 and NB4-LR2 cells. The three cell lines express


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

10

similar levels of the catalytic PKA-C subunit (Figure 1A and B). Treatment with a moderate

concentration (100 µM) of the pan-PKA activator, cAMP analog, 8-CPT-cAMP did not modify

significantly c-Myc protein level in either the NB4 parental cells or its ATRA-maturation

resistant NB4-LR2 subline. In contrast, c-Myc was almost completely depleted in the NB4-

LR1SFD cells after 48h of treatment with the cAMP analog. Note that 8-CPT-cAMP induced a

significant increase of PKA-RI level in both NB4wt and NB4-LR2 cells. A similar c-AMP-

mediated increase has already been reported in other cellular models and attributed to the

presence of a consensus cAMP response element (CRE) as well as several AP-2 sites in the

5’-upstream region of the RIα gene (Taskén et al., 1991; Solberg et al., 1997).

The depletion of c-Myc was apparent already after 6h of treatment (Figure 2A). To probe

whether the effect of 8-CPT-cAMP treatment is transcriptional, we evaluated changes in c-

Myc mRNA levels by qRT-PCR (Figure 2B). We found that, after an initial and transient

decrease, the c-Myc mRNA level stabilized at about 80% of the level in control cells. Thus,

after 48 h of treatment with cAMP analog the c-Myc mRNA was still robustly expressed while

c-Myc remained hardly detectable at the protein level. This suggests that the 8-CPT-cAMP-

induced c-Myc decrease results from a combination of modest transcriptional and strong

post-transcriptional effects. Because the 26S proteasome has been incriminated in c-Myc

degradation (Sears, 2004), we exposed NB4-LR1SFD cells to 100 µM of 8-CPT-cAMP for 6h

(Figure 2C) in the presence or absence of the proteasome inhibitor MG132. We found that

the NB4-LR1SFD cells treated with MG132 had increased Myc protein level and maintained a

high level of c-Myc even in the presence of 8-CPT-cAMP. This indicates that the decrease of

c-Myc induced by 8-CPT-cAMP in NB4-LR1SFD cells is strongly proteasome dependent. Of

note, a transcriptional role of MG132 cannot be excluded since the presence of MG132 partly

abolished the initial decrease of c-Myc mRNA level induced by 8-CPT-cAMP treatment

(Figure 2D).


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

11

Disruption of both PKA-RII isozymes is necessary for 8-CPT-cAMP induced c-Myc

proteasomal degradation.

That 8-CPT-cAMP-induced c-Myc decrease in NB4-LR1SFD cells, but not in the parental NB4

cells nor in the ATRA-maturation resistant NB4-LR2 cells (Figure 1), suggests that its effect

might require a high cellular PKA-I/PKA-II ratio. In order to probe the respective involvement

of PKA-I and PKA-II isozymes we used, at first, RNA interference technology to knockdown

the PKA regulatory subunits in the NB4 cell line and produce NB4-RIIαKD or NB4-RIIβKD

stable single knockdown and NB4-RIIα,βKD stable double knockdown sublines. Western blot

analyses confirmed the specific loss of PKA-RIIα and PKA-RIIβ in NB4-RIIαKD and NB4-

RIIβKD cells, respectively (Figure 3A and B). The NB4-RIIα,βKD cells were previously

described (Gausdal et al., 2013). They are characterized by an almost complete lack of both

PKA RII subunits, which is compensated by a higher level of RI subunit. The consequence is

an increased PKAI/PKAII ratio compared to NB4 control and single knockdown cells. Hence,

the NB4-RIIα,βKD cells mimicked NB4-LR1SFD cells regarding the PKA isozyme background.

The NB4-RIIα,βKD cells mimicked also these cells regarding the strong lowering of c-Myc

protein level after exposure to 8-CPT-cAMP, although higher 8-CPT-cAMP concentration

(200 µM) was required to obtain near full depletion of c-Myc (Figure 3). At this level of 8-

CPT-cAMP a modest (≤ 30%) decrease of c-Myc was noted also in some of the PKA-RII

expressing cell lines (Figure 3). Of note, as in NB4-LR1SFD cells (Figure 2A), in NB4-

RIIα,βKD cells, 8-CPT-cAMP-induced destabilisation of c-Myc protein was already observed

after 6h of treatment (Supplementary Figure 1). Altogether, the data obtained on both NB4-

LR1SFD and NB4 knockdown cells support the notion that PKA-RII is important for c-Myc

stability.

Activation of PKA-I decreases c-Myc stability only in PKA-II deficient NB4 cell lines.

We did take advantage of the availability of specific PKA I agonists and antagonists to

address the role of PKA-I in c-Myc stability. Indeed, we exploited the fact that the


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

12

combination of site-specific cAMP analogs achieved preferential activation of PKA-I (Huseby

et al., 2011; Ogreid et al., 1989). Therefore, the site AI-selective analog (N6-Bz-8-piperidino-

cAMP) was combined with the site BI-preferring analog (2-Cl-8-AHA-cAMP) to achieve

preferential activation of PKA-I. Both NB4-LR1SFD and NB4RIIα,βKD cells showed similar

dose-dependent decrease in the relative level of c-Myc protein, with about 70% decrease

after 6h (Figure 4 A and B). The NB4 parental or NB4 scramble control cells showed much

less decrease (Figure 4 C and D). These results indicate that the activation of PKA-I is able

to strongly decrease c-Myc stability only in a PKA-II deficiency context. This conclusion is

supported by the ability of the PKA-I-preferring competitive inhibitor Rp-8-Br-cAMPS

(Gjertsen et al. 1995) to counteract the effect of PKA-I activation (Figure 4 E).

The pharmacological c-Myc inhibitor 10058-F4 is less efficient than 8-CPT-cAMP to

increase C/EBPβ and drive ATRA-induced NB4-LR1SFD cell maturation.

NB4-LR1SFD cells require, like NB4-LR1 cells, the exogenous stimulation of cAMP signaling

in addition to ATRA treatment for maturation (Duprez et al., 2000; Lanotte et al., 1991;

Pendino et al., 2002). The induction of CCAAT/enhancer binding protein β (C/EBPβ) is

shown as an early critical mediator of ATRA-mediated granulocytic differentiation in APL

cells (D'Alo et al., 2003; Duprez et al., 2003; Truong et al., 2003). Because c-Myc is known to

inhibit cell differentiation through C/EBPβ (Duprez et al., 2003; Mink et al., 1996; Pan et al.,

2014), we wanted to know whether the pharmacological inhibition of c-Myc was sufficient to

replace the effect of 8-CPT-cAMP to restore NB4-LR1SFD cell differentiation and C/EBPβ

induction by ATRA. For this we used the 10058-F4 c-Myc inhibitor small molecule. Although

originally described to disrupt mainly the c-Myc/Max heterodimerization (Follis et al., 2009),

this inhibitor decreases c-Myc protein level and induces myeloid differentiation in AML cells

(Pan et al. 2014, Huang et al.2006, Lin et al. 2007, Zirath et al 2013). These two properties

constitute the rational basis for using this compound to investigate the role of c-Myc protein

in PKA-mediated differentiation. First, we treated the cells with 10058-F4 in the presence or


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

13

absence of ATRA and examined the induction of maturation by the NBT reduction assay and

the expression of the maturation specific markers: the cell surface marker, CD11c, and the

transcription factor, C/EBPβ. Figure 5A shows no NBT positive cells when cells were treated

with 10058-F4 alone or together with ATRA. However, a slight but significant increase of

CD11c and C/EBPβ was observed in cells treated with the combination of 10058-F4 and

ATRA (Figure 5B and C). In contrast, cells treated with ATRA plus 8-CPT-cAMP were able to

reduce NBT to blue formazan as expected for a treatment inducing terminal granulocytic

differentiation. Moreover, the cells exhibited a much higher CD11c and C/EBPβ levels when

co-treated with ATRA and 8-CPT-cAMP compared to the co-treatment with ATRA and

10058-F4 (Figure 5B and C). Therefore, the inhibition of c-Myc combined with ATRA

treatment is not sufficient to induce APL cell progression towards a fully differentiated state

demonstrating the importance of additional PKA dependent factors.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

14

DISCUSSION The present study reveals novel aspects of the interacting role of cAMP effectors and c-Myc

in retinoid APL differentiation. Using NB4 variant APL sublines that differ with respect to PKA

isoform expression, we demonstrate that PKA activation is able to decrease the level of c-

Myc protein. In a recent paper, Padmanabhan et al. (Padmanabhan et al., 2013) reported

that a brief pharmacological pan-PKA inhibition using H89, a small molecule marketed as a

PKA inhibitor, diminished c-Myc level through a post-translational mechanism mediated by

the proteasome, whereas PKA activation with forskolin induced c-Myc protein accumulation.

They showed also that a prolonged PKACα knockdown increased c-Myc through a

transcriptional mechanism, suggesting that short and long term effects on PKA activity may

lead to different responses. The apparent discrepancy observed between our data in NB4-

LR1SFD and NB4-RIIα,βKD with the data reported by Padmanabhan et al. in a prostate

cancer cell line (PC3) may be due to a difference in the specificity of the drugs used to

activate or inhibit PKA, in particular the H89 drug. Indeed, some caution needs to be taken

when interpreting the data utilizing this drug extensively used as a PKA inhibitor, since it

inhibits also at least 8 other kinases including the RhoA/ROCK (Davies et al., 2000; Lochner

and Moolman, 2006). This is relevant since PKA inhibits the activation of Rho kinase

(Leemhuis et al., 2002; Petersen et al., 2008).

Alternatively, this divergence may reflect a difference in PKA-I/PKA-II ratio between the cell

lines studied. Interestingly, we observed the PKA-dependent decrease of c-Myc stability

mainly in a PKA-II deficient background (such as that present in either NB4-LR1SFD or NB4

cells with knockdown of both PKA-RIIα and PKA-RIIβ). Using selective PKA-I agonists we

show that activation of PKA-I recapitulates the effects of the pan-PKA agonist 8-CPT-cAMP

on c-Myc expression, supporting the notion that its effect is mediated solely through PKA-I

activation. The PKA-II isozyme appears to have a protective role against c-Myc degradation.

Indeed, 8-CPT-cAMP decreased c-Myc stability much more strongly in the PKA-II deficient

NB4-LR1SFD cells than in NB4 cells (expressing both PKA-I and PKA-II). The present study


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

15

further reinforces the notion that distinct cellular responses to cAMP may be elicited upon

selective activation of one isozyme and that the cAMP response can be influenced by PKA

isozyme switching, providing a new way in which PKA can control c-Myc protein stability.

Although the major mechanism by which PKA-I lowers c-Myc appears to be proteasome-

dependent based on the studies with proteasome inhibitor, a transcriptional contribution by

PKA-I signalling on c-Myc expression cannot be excluded. Indeed RIα subunit has been

shown to interact with a transcriptional repressor of c-Myc, PATZ1 (Fedele et al., 2000),

whose nuclear translocation is inducible by cAMP (Yang et al., 2010).

c-Myc is aberrantly expressed in a wide variety of human solid tumors as well as leukemia

where its expression is closely related to chemotherapy resistance (Delgado et al., 2013;

Knapp et al., 2003; Leonetti et al., 1999). Differentiation is accompanied by downregulation

of c-Myc, whose ectopic expression blocks terminal differentiation in various cellular models

(Coppola and Cole, 1986; Larsson et al., 1988; Gomez-Casares et al., 2013), including the

ATRA-induced NB4 cell differentiation model used in the present study (Pan et al., 2014).

NB4-LR1SFD cells treated with 10058-F4 combined with ATRA underwent a strong decrease

of c-Myc protein. The same treatment failed to induce terminal NB4-LR1SFD cell

differentiation, although it did induce some of the molecular modifications (c-Myc decrease

and a moderate C/EBPß and CD11c increase) triggered by 8-CPT-cAMP and ATRA

cotreatment. We propose that c-Myc downregulation may have a permissive role to drive

maturation of the ATRA-maturation resistant NB4-LR1SFD cells, one mechanism being the

relief of the tonic inhibition by c-Myc on C/EBPß expression. Our findings indicate that

cAMP/PKA-dependent signalling remains essential, in addition to c-Myc downregulation, for

maturation of these cells.

Our results could have general clinical implications. Resistance to ATRA-induced

differentiation is the main reason for relapse in APL disease, but also for the non-efficacy of

ATRA treatment in other forms of leukemia. Many AML-derived cell lines are sensitive to

cAMP-triggered differentiation (Benoit et al., 2001; Ruchaud et al., 1994). Our findings show


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

16

(1) that PKA activation leads to decreased c-Myc protein stability in the ATRA-maturation

resistant NB4-LR1SFD cells, and (2) that this decrease is important for the commitment to the

differentiation process, but not sufficient to achieve overt terminal differentiation, thus

providing a likely mechanism by which cAMP elevating agents can restore ATRA-induced

differentiation in maturation-resistant APL cells. If cAMP-PKA based therapy is to be

considered in the clinical management of APL or other leukemia, a special attention should

be given to the PKA-I/PKA-II ratio. Furthermore, inhibition of c-Myc activity should also be

considered in combined therapies to overcome ATRA resistance.

Aknowledgments

The authors thank Jacqueline Varennes (UMR-S 1007) for her technical assistance in

Western blot and Philippe Bardy (Centre de Langues, Université Paris Descartes) for English

editing of the paper.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

17

Authorship Contributions

Participated in Research Design: Qingyuan Liu, Eric Nguyen, Stein Døskeland, Evelyne

Ségal-Bendirdjian

Conducted Experiments: Qingyuan Liu, Eric Nguyen

Wrote or contributed to the writing of the manuscript: Qingyuan Liu, Eric Nguyen, Stein

Døskeland, Evelyne Ségal-Bendirdjian


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

18

REFERENCES

Benoit G, Roussel M, Pendino F, Segal-Bendirdjian E, and Lanotte M (2001) Orchestration of

multiple arrays of signal cross-talk and combinatorial interactions for maturation and

cell death: another vision of t(15;17) preleukemic blast and APL-cell maturation.

Oncogene 20: 7161-7177.

Bernard S and Eilers M (2006) Control of cell proliferation and growth by Myc proteins.

Results Probl Cell Differ 42: 329-342.

Cho-Chung YS, Pepe S, Clair T, Budillon A, and Nesterova M (1995) cAMP-dependent

protein kinase: role in normal and malignant growth. Crit Rev Oncol Hematol 21: 33-

61.

Coppola JA and Cole MD (1986) Constitutive c-myc oncogene expression blocks mouse

erythroleukaemia cell differentiation but not commitment. Nature 320: 760-763.

D'Alo F, Johansen LM, Nelson EA, Radomska HS, Evans EK, Zhang P, Nerlov C, and Tenen

DG (2003) The amino terminal and E2F interaction domains are critical for C/EBP

alpha-mediated induction of granulopoietic development of hematopoietic cells. Blood

102: 3163-3171.

Dang CV (1999) c-Myc target genes involved in cell growth, apoptosis, and metabolism. Mol

Cell Biol 19: 1-11.

Dang CV, Le A, and Gao P (2009) MYC-induced cancer cell energy metabolism and

therapeutic opportunities. Clin Cancer Res 15: 6479-6483.

Dang CV, O'Donnell KA, Zeller KI, Nguyen T, Osthus RC, and Li F (2006) The c-Myc target

gene network. Semin Cancer Biol 16: 253-264.

Dani C, Blanchard JM, Piechaczyk M, El Sabouty S, Marty L, and Jeanteur P (1984) Extreme

instability of myc mRNA in normal and transformed human cells. Proc Natl Acad Sci

U S A 81: 7046-7050.

Davies SP, Reddy H, Caivano M, and Cohen P (2000) Specificity and mechanism of action

of some commonly used protein kinase inhibitors. Biochem J 351: 95-105.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

19

Delgado MD, Albajar M, Gomez-Casares MT, Batlle A, and Leon J (2013) MYC oncogene in

myeloid neoplasias. Clin Transl Oncol 15: 87-94.

Deville L, Hillion J, Pendino F, Samy M, Nguyen E, and Segal-Bendirdjian E (2011) hTERT

promotes imatinib resistance in chronic myeloid leukemia cells: therapeutic

implications. Mol Cancer Ther 10: 711-719.

Duprez E, Benoit G, Flexor M, Lillehaug JR, and Lanotte M (2000) A mutated PML/RARA

found in the retinoid maturation resistant NB4 subclone, NB4-R2, blocks RARA and

wild-type PML/RARA transcriptional activities. Leukemia 14: 255-261.

Duprez E, Wagner K, Koch H, and Tenen DG (2003) C/EBPbeta: a major PML-RARA-

responsive gene in retinoic acid-induced differentiation of APL cells. Embo J 22:

5806-5816.

Fedele M, Benvenuto G, Pero R, Majello B, Battista S, Lembo F, Vollono E, Day PM,

Santoro M, Lania L, Bruni CB, Fusco A, and Chiariotti L (2000) A novel member of

the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts

as a transcriptional repressor. J Biol Chem 275: 7894-7901.

Follis AV, Hammoudeh DI, Daab AT, and Metallo SJ (2009) Small-molecule perturbation of

competing interactions between c-Myc and Max. Bioorg Med Chem Lett 19: 807-810.

Gausdal G, Wergeland A, Skavland J, Nguyen E, Pendino F, Rouhee N, McCormack E,

Herfindal L, Kleppe R, Havemann U, Schwede F, Bruserud O, Gjertsen BT, Lanotte

M, Segal-Bendirdjian E, and Doskeland SO (2013) Cyclic AMP can promote APL

progression and protect myeloid leukemia cells against anthracycline-induced

apoptosis. Cell Death Dis 4: e516.

Gjertsen BT, Mellgren G, Otten A, Maronde,E, Genieser HG, Jastorff B, Vintermyr OK,

McKnight GS, and Doskeland SO (1995) Novel (Rp)-cAMPS analogs as tools for

inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity modulates

interleukin-1 beta action. J Biol Chem 270: 20599-20607.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

20

Gomez-Casares MT, Garcia-Alegria E, Lopez-Jorge CE, Ferrandiz N, Blanco R, Alvarez S,

Vaque JP, Bretones G, Caraballo JM, Sanchez-Bailon P, Delgado MD, Martin-Perez

J, Cigudosa JC, and Leon J (2013) MYC antagonizes the differentiation induced by

imatinib in chronic myeloid leukemia cells through downregulation of p27(KIP1.).

Oncogene 32: 2239-2246.

Greider CW (1999) Telomerase activation. One step on the road to cancer? Trends Genet

15: 109-112.

Guillemin MC, Raffoux E, Vitoux D, Kogan S, Soilihi H, Lallemand-Breitenbach V, Zhu J,

Janin A, Daniel MT, Gourmel B, Degos L, Dombret H, Lanotte M, and De The H

(2002) In vivo activation of cAMP signaling induces growth arrest and differentiation

in acute promyelocytic leukemia. J Exp Med 196: 1373-1380.

Hoffman B and Liebermann DA (2008) Apoptotic signaling by c-MYC. Oncogene 27: 6462-

6472.

Huang MJ, Cheng YC, Liu C-R, Lin S, and Liu HE (2006) A small-molecule c-Myc inhibitor,

10058-F4, induces cell-cycle arrest, apoptosis, and myeloid differentiation of human

acute myeloid leukemia Exp Hematol 34: 1480–1489

Huseby S, Gausdal G, Keen TJ, Kjaerland E, Krakstad C, Myhren L, Bronstad K, Kunick C,

Schwede F, Genieser HG, Kleppe R, and Doskeland SO (2011) Cyclic AMP induces

IPC leukemia cell apoptosis via CRE-and CDK-dependent Bim transcription. Cell

Death Dis 2: e237.

Ji Z, Mei FC, Miller AL, Thompson EB, and Cheng X (2008) Protein kinase A (PKA) isoform

RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute

lymphoblastic leukemia cells. J Biol Chem 283: 21920-21925.

Jiang G, Albihn A, Tang T, Tian Z, and Henriksson M (2008) Role of Myc in differentiation

and apoptosis in HL60 cells after exposure to arsenic trioxide or all-trans retinoic acid.

Leuk Res 32: 297-307.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

21

Kamashev D, Vitoux D, and De The H (2004) PML-RARA-RXR oligomers mediate retinoid

and rexinoid/cAMP cross-talk in acute promyelocytic leukemia cell differentiation. J

Exp Med 199: 1163-1174.

Knapp DC, Mata JE, Reddy MT, Devi GR, and Iversen PL (2003) Resistance to

chemotherapeutic drugs overcome by c-Myc inhibition in a Lewis lung carcinoma

murine model. Anticancer Drugs 14: 39-47.

Kopperud R, Krakstad C, Selheim F, and Doskeland SO (2003) cAMP effector mechanisms.

Novel twists for an 'old' signaling system. FEBS Lett 546: 121-126.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 227: 680-685.

Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, and Berger R (1991) NB4,

a maturation inducible cell line with t(15;17) marker isolated from a human acute

promyelocytic leukemia (M3). Blood 77: 1080-1086.

Larsson LG, Ivhed I, Gidlund M, Pettersson U, Vennstrom B, and Nilsson K (1988) Phorbol

ester-induced terminal differentiation is inhibited in human U-937 monoblastic cells

expressing a v-myc oncogene. Proc Natl Acad Sci U S A 85: 2638-2642.

Leemhuis J, Boutillier S, Schmidt G, and Meyer DK (2002) The protein kinase A inhibitor H89

acts on cell morphology by inhibiting Rho kinase. J Pharmacol Exp Ther 300: 1000-

1007.

Lemm I and Ross J (2002) Regulation of c-myc mRNA decay by translational pausing in a

coding region instability determinant. Mol Cell Biol 22: 3959-3969.

Leonetti C, Biroccio A, Candiloro A, Citro G, Fornari C, Mottolese M, Del Bufalo D, and Zupi

G (1999) Increase of cisplatin sensitivity by c-myc antisense oligodeoxynucleotides in

a human metastatic melanoma inherently resistant to cisplatin. Clin Cancer Res 5:

2588-2595.

Lin CP, Liu JD, Chow JM, Liu CR, and Liu HE. (2007) Small-molecule c-Myc inhibitor, 10058-

F4, inhibits proliferation, downregulates human telomerase reverse transcriptase and


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

22

enhances chemosensitivity in human hepatocellular carcinoma cells. Anticancer

Drugs 18: 161–170;

Lochner A and Moolman JA (2006) The many faces of H89: a review. Cardiovasc Drug Rev

24: 261-274.

Malempati S, Tibbitts D, Cunningham M, Akkari Y, Olson S, Fan G, and Sears RC (2006)

Aberrant stabilization of c-Myc protein in some lymphoblastic leukemias. Leukemia

20: 1572-1581.

Mink S, Mutschler B, Weiskirchen R, Bister K, and Klempnauer KH (1996) A novel function

for Myc: inhibition of C/EBP-dependent gene activation. Proc Natl Acad Sci U S A 93:

6635-6640.

Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, Piqani B, Eisenhaure TM,

Luo B, Grenier JK, Carpenter AE, Foo SY, Stewart SA, Stockwell BR, Hacohen N,

Hahn WC, Lander ES, Sabatini DM, and Root DE (2006) A lentiviral RNAi library for

human and mouse genes applied to an arrayed viral high-content screen. Cell 124:

1283-1298.

Nguyen E, Gausdal G, Varennes J, Pendino F, Lanotte M, Doskeland SO, and Segal-

Bendirdjian E (2013) Activation of both protein kinase A (PKA) type I and PKA type II

isozymes is required for retinoid-induced maturation of acute promyelocytic leukemia

cells. Mol Pharmacol 83: 1057-1065.

Ogreid D, Ekanger R, Suva RH, Miller JP, and Doskeland SO (1989) Comparison of the two

classes of binding sites (A and B) of type I and type II cyclic-AMP-dependent protein

kinases by using cyclic nucleotide analogs. Eur J Biochem 181: 19-31.

Oh S, Song YH, Kim UJ, Yim J, and Kim TK (1999) In vivo and in vitro analyses of Myc for

differential promoter activities of the human telomerase (hTERT) gene in normal and

tumor cells. Biochem Biophys Res Commun 263: 361-365.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

23

Padmanabhan A, Li X, and Bieberich CJ (2013) Protein kinase A regulates MYC protein

through transcriptional and post-translational mechanisms in a catalytic subunit

isoform-specific manner. J Biol Chem 288: 14158-14169.

Pan XN, Chen JJ, Wang LX, Xiao RZ, Liu LL, Fang ZG, Liu Q, Long ZJ, and Lin DJ (2014)

Inhibition of c-Myc overcomes cytotoxic drug resistance in acute myeloid leukemia

cells by promoting differentiation. PLoS One 9: e105381.

Pendino F, Sahraoui T, Lanotte M, and Segal-Bendirdjian E (2002) A novel mechanism of

retinoic acid resistance in acute promyelocytic leukemia cells through a defective

pathway in telomerase regulation. Leukemia 16: 826-832.

Petersen RK, Madsen L, Pedersen LM, Hallenborg P, Hagland H, Viste K, Doskeland SO,

and Kristiansen K (2008) Cyclic AMP (cAMP)-mediated stimulation of adipocyte

differentiation requires the synergistic action of Epac- and cAMP-dependent protein

kinase-dependent processes. Mol Cell Biol 28: 3804-3816.

Pick E (1986) Microassays for superoxide and hydrogen peroxide production and nitroblue

tetrazolium reduction using an enzyme immunoassay microplate reader. Methods

Enzymol 132: 407-421.

Pidoux G and Tasken K (2010) Specificity and spatial dynamics of protein kinase A signaling

organized by A-kinase-anchoring proteins. J Mol Endocrinol 44: 271-284.

Quenech'Du N, Ruchaud S, Khelef N, Guiso N, and Lanotte M (1998) A sustained increase

in the endogenous level of cAMP reduces the retinoid concentration required for APL

cell maturation to near physiological levels. Leukemia 12: 1829-1833.

Rohlff C, Clair T, and Cho-Chung YS (1993) 8-Cl-cAMP induces truncation and down-

regulation of the RI alpha subunit and up-regulation of the RII beta subunit of cAMP-

dependent protein kinase leading to type II holoenzyme-dependent growth inhibition

and differentiation of HL-60 leukemia cells. J Biol Chem 268: 5774-5782.

Ruchaud S, Duprez E, Gendron MC, Houge G, Genieser HG, Jastorff B, Doskeland SO, and

Lanotte M (1994) Two distinctly regulated events, priming and triggering, during


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

24

retinoid-induced maturation and resistance of NB4 promyelocytic leukemia cell line.

Proc Natl Acad Sci U S A 91: 8428-8432.

Sarbassov DD, Guertin DA, Ali SM, and Sabatini DM (2005) Phosphorylation and regulation

of Akt/PKB by the rictor-mTOR complex. Science 307: 1098-1101.

Schmidt EV (1999) The role of c-myc in cellular growth control. Oncogene 18: 2988-2996.

Schwartz DA and Rubin CS (1985) Identification and differential expression of two forms of

regulatory subunits (RII) of cAMP-dependent protein kinase II in Friend

erythroleukemic cells. Differentiation and 8-bromo-cAMP elicit a large and selective

increase in the rate of biosynthesis of only one type of RII. J Biol Chem 260: 6296-

62303.

Sears RC (2004) The life cycle of C-myc: from synthesis to degradation. Cell Cycle 3: 1133-

1137.

Solberg R, Sandberg M, Natarajan V, Torjesen P, Hansson V, Jahnsen T, and Taskén KA

(1997) The human gene for the regulatory subunit RIa of cyclic adenosine 3',5'-

monophosphate-dependent protein kinase: two distinct promoters provide differential

regulation of alternately spliced messenger ribonucleic acids. Endocrinology 138:

169-181.

Taskén KA, Knutsen HK, Attramadal H, Taskén K, Jahnsen T, Hansson V, and Eskild W

(1991) Different mechanisms are involved in cAMP-mediated induction of mRNAs for

subunits of cAMP-dependent protein kinases. Mol Endocrinol 5: 21-28.

Taylor SS, Zhang P, Steichen JM, Keshwani MM, and Kornev AP (2013) PKA: lessons

learned after twenty years. Biochim Biophys Acta 1834: 1271-1278.

Truong BT, Lee YJ, Lodie TA, Park DJ, Perrotti D, Watanabe N, Koeffler HP, Nakajima H,

Tenen DG, and Kogan SC (2003) CCAAT/Enhancer binding proteins repress the

leukemic phenotype of acute myeloid leukemia. Blood 101: 1141-1148.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

25

Wall M, Poortinga G, Hannan KM, Pearson RB, Hannan RD, and McArthur GA (2008)

Translational control of c-MYC by rapamycin promotes terminal myeloid

differentiation. Blood 112: 2305-2317.

Wu KJ, Grandori C, Amacker M, Simon-Vermot N, Polack A, Lingner J, and Dalla-Favera R

(1999) Direct activation of TERT transcription by c-MYC. Nat Genet 21: 220-4.

Yang WL, Ravatn R, Kudoh K, Alabanza L, and Chin KV (2010) Interaction of the regulatory

subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278). Biochem

Biophys Res Commun 391: 1318-1323.

Zirath H, Frenzel A. Oliynyk G, Segerström L, Westermark UK, Larsson K, Munksgaard

Persson M, Hultenby K, Lehtiö J, Einvik C, Påhlman S, Kogner P, Jakobsson PJ, and

Henriksson MA (2013) MYC inhibition induces metabolic changes leading to

accumulation of lipid droplets in tumor cells, Proc Natl Acad Sci USA 110: 10258–

10263.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

26

Footnotes

Financial supports: This work was supported by grants from the Institut National de la Santé

et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique

(CNRS), the Fondation de France (E. S-B.), the Ligue Nationale Contre le Cancer (Comité Ile

de France, E. S-B.), and fellowship from China Scholarship Council (QY L.). SD has received

financial support from the Norwegian Cancer Society and the West Norw. Health Authorities.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

27

Figures legends

Figure 1: c-Myc protein level decreases dramatically upon 8-CPT-cAMP treatment in

NB4-LR1SFDcells not in the NB4 and NB4-LR2 cells. NB4, NB4-LR2 and NB4-LR1SFD cells

were treated with 8-CPT-cAMP (100µM) for 48 h. A. Expression of c-Myc and PKA subunits

(RI, RIIα, RIIβ, and C) were analysed by Western blot. Lamin B was used as a loading

control. The images are representative of results obtained from independent experiments

performed three to five times. B. For each protein detected in A, band intensities were

quantified by densitometry, normalized relatively to lamin B signals and represented as

percentage of levels in the NB4 control cells. PKA-RI/PKA-RII expressed the ratio of PKA-RI

to PKA-RIIα+PKA-RIIβ band intensities. Bars in each panel represent means with error bars

corresponding to S.E.M. ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 2: 8-CPT-cAMP induces a proteasomal degradation of c-Myc in the maturation

resistant NB4-LR1SFD cells. NB4-LR1SFD cells were treated with 8-CPT-cAMP (100µM) for

the indicated time. A. Upper panel: Western blots showing that 8-CPT-cAMP (100 µM)

treatment decreases c-Myc protein levels in NB4-LR1SFD cells. Lower panel: c-Myc signal

was quantified by densitometry, normalized relatively to lamin B signals and represented as

percentage of levels in the NB4-LR1SFD control cells. B. Quantitative RT-PCR showing a

transient decrease of c-Myc mRNA in the NB4-LR1SFD cell line. c-Myc mRNA was normalized

to the expression of GAPDH. C. Cells are treated with 8-CPT-cAMP (100 µM) for 6h in the

absence or in the presence of MG132. Upper panel: Western blots showing that proteasome

inhibition with 2.5 µM proteasome inhibitor MG132 prevents the strong decrease of c-Myc

protein. Lower panel: c-Myc signal was quantified by densitometry, normalized relatively to

lamin B signals and represented as percentage of levels in the NB4-LR1SFD control cells. D.

Quantitative RT-PCR. c-Myc mRNA was normalized to the expression of GAPDH. The

images in A and C are representative of results obtained from independent experiments


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

28

performed three times. Bars in each panel represent means with error bars corresponding to

S.E.M. * p<0.05, ** p<0.01, *** p<0.001.

Figure 3: Knock down of both PKA-RII isozymes is necessary for 8-CPT-cAMP induced

c-Myc proteasomal degradation in NB4 cells. A. NB4wt, NB4scr, NB4-RIIαKD, NB4-

RIIβKD and NB4-RIIα,βKD cells were cultured for 48 h in the absence or presence of 8-CPT-

cAMP (200 µM). Expression of c-Myc and PKA subunits (RIα, RIIα, and RIIβ) were analysed

by Western blot. The images are representative of results obtained from independent

experiments performed three to five times. B. For each protein detected in A, signals were

quantified by densitometry, normalized relatively to lamin B signals and represented as

percentage of levels in the NB4 control cells. PKA-RI/PKA-RII expressed the ratio of signals

obtained measuring PKA-RI to PKA-RIIα+PKA-RIIβ signals. Bars in each panel represent

means with error bars corresponding to S.E.M. * p<0.05, ** p<0.01, *** p<0.001, ****

p<0.0001.

Figure 4: Activation of PKA-I alone is able to decrease c-Myc stability only in a PKA-II

deficient cells. NB4-LR1SFD (A), NB4-RIIα,βKD (B), NB4wt (C), and NB4scr (D) cells were

cultured for 6 h in the absence or presence of increasing concentrations of a combination of

two cAMP analogs (2Cl-8-AHA-cAMP and N6-Bz-8-Pip-cAMP) known to activate PKA-I and

not PKA-II. The respective concentration of each analog is indicated. Expression of c-Myc

was analysed by Western blot. E. NB4-LR1SFD cells were cultured for 6 h in the absence or

presence of 8-CPT-CAMP (100 µM) alone or in combination with the PKA-I antagonist Rp-8-

Br-cAMP (1 mM, added 30 min before 8-CPT-cAMP addition). Expression of c-Myc was

analysed by Western blot. In all experiments, Lamin B was used as a loading control. c-Myc

signals were normalized to Lamin B and represented as percentage of c-Myc levels in the

control cells. The images are representative of results obtained from independent


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


MOL # 97915

29

experiments performed three times. Bars in each panel represent means with error bars

corresponding to S.E.M. * p<0.05, ** p<0.01, *** p<0.001.

Figure 5. The pharmacological decrease of c-Myc by 10058-F4 is less efficient than 8-

CPT-cAMP to increase C/EBPβ and drive ATRA-induced NB4-LR1SFD cell maturation.

NB4-LR1SFD cells were treated with ATRA (1 µM), 10058-F4 (50 µM) or 8-CPT-cAMP (100

µM) alone or in combination. ATRA (1 µM) + 8-CPT-cAMP (100 µM) treatment was

performed as a positive control for the induction of terminal granulocytic differentiation. A.

NBT assay was performed after 72 h of treatment. B. Western blotting analysis was

performed after 48 h of treatment to detect expression of c-Myc, CEBPβ, and CD11c.

GAPDH was used as a loading control. The images are representative of results obtained

from three independent experiments. C. Protein band signals were quantified by

densitometry, normalized relatively to GAPDH signals and represented as percentage of

levels in the NB4-LR1SFD control untreated cells (c-Myc) or in the fully differentiated NB4-

LR1SFD ATRA+8-CPT-cAMP treated cells (C/EBPβ and CD11c). Bars in each panel

represent means with error bars corresponding to S.E.M. * p<0.05, ** p<0.01.


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


Figure 1

A

PK

A-R

IIa

pro

tein

rela

tive

le

ve

l

0

0.5

1.0

1.5

0

1.0

2.0

PK

A-C

pro

tein

rela

tive

le

ve

l

B

8-CPT-cAMP control

c-M

yc p

rote

in

rela

tive

le

ve

l

1.0

2.0

****

0

1.0

2.0

3.0

4.0

PK

A-R

I p

rote

in

rela

tive

le

ve

l

**

***

****

0

1.0

2.0

3.0

PK

A-R

IIb

pro

tein

rela

tive

le

ve

l

****

PK

A R

I/P

KA

RII

rela

tive

le

ve

l

*** ***

0

0.05

0.10

0.15 5

10

15


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


Figure 2

c-Myc

Lamin B

NB4-LR1SFD

0 6 24 48

8-CPT-cAMP (100 µM)

Time (h)

A

c-Myc

Lamin B

8-CPT-cAMP

MG132

C NB4-LR1SFD

- -

+ + -

- + +

0 6 24 48 96

c-M

yc m

RN

A

rela

tive

leve

l

c-M

yc m

RN

A

rela

tive

leve

l

Time (h) 0

20

40

60

80

100

120

0

20

40

60

80

100

120

8-CPT-cAMP (6 h)

MG132 (6 h)

c-M

yc p

rote

in

rela

tive

le

ve

l

0

0.2

0.4

0.6

0.8

1.0

c-M

yc p

rote

in

rela

tive

le

ve

l

0

1

2

3

4

5

6

B D

*

**

* ***

*** ***

**

ns


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


Figure 3

A

c-Myc

PKA RIIa

PKA RIIb

Lamin B

Time (h)

8-CPT-cAMP (200 µM)

NB4wt NB4scr NB4RIIaKD

C 48 C 48 C 48 C 48

NB4RIIbKD

C 48

NB4RIIa,bKD

B

PKA RI

0

0.2

0.4

0.6

0.8

1.0

PK

A R

IIa

pro

tein

rela

tive

le

ve

l

8-CPT-cAMP

control

** *

PK

A R

I/P

KA

RII

re

lative

le

ve

l

PK

A R

I p

rote

in

rela

tive

le

ve

l

10

20

30

40

**

****

* **

*

50

PK

A R

IIb

pro

tein

rela

tive

le

ve

l

0

0.2

0.4

0.6

0.8

1.0

1.2

* * 1.4

c-M

yc p

rote

in

rela

tive

le

ve

l

0

0.2

0.4

0.6

0.8

1.0 **

* ***

** 1.2

1.2

**

*

**

*** ***

**

**

0

0.5

1.0

1.5

4

6

8

*


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


NB4RIIa,bKD

0 15;45 30;90 45;135

2Cl8AHA; N6BZ8PIP

NB4-LR1SFD

PKA-I

0 15;45 30;90 45;135

2Cl8AHA; N6BZ8PIP

PKA-I

µM

c-Myc

Lamin B

A B

8-CPT-cAMP

Rp-8-Br-cAMPS

- -

+ + -

- + +

NB4-LR1SFD

c-Myc

Lamin B

E

0 15;45 30;90 45;135

2Cl8AHA; N6BZ8PIP

NB4wt

PKA-I

0 15;45 30;90 45;135

2Cl8AHA; N6BZ8PIP

PKA-I

NB4scr D C

c-M

yc p

roe

tin

rela

tive

le

ve

l

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

µM

c-Myc

Lamin B

Figure 4

**

** *** * **

* ns

c-M

yc p

roe

tin

rela

tive

le

ve

l

c-M

yc p

roe

tin

rela

tive

leve

l


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


Figure 5

NB4-LR1SFD

A 10058-F4

(50 µM) 8-CPT-cAMP

(100 µM) -

ATRA

(1 µM)

+

-

8-CPT-cAMP (C) 100 µM

NB4-LR1SFD

10058-F4 (I) 50 µM

ATRA (A) 1 µM

- - -

- -

+

- +

-

+ - +

- -

+

+ + -

c-Myc

CEBPβ

GAPDH

B

CD11c

0

0.2

0.4

0.6

0.8

1.0

c-M

yc p

rte

in

rela

tive

le

ve

l

0

0.2

0.4

0.6

0.8

1.0

CE

BPβ

pro

etin

rela

tive

le

ve

l

CD

11

c p

roe

tin

rela

tive

le

ve

l

C

0 0.02 0.04 0.06 0.08 0.8

1

- I C A A+I A+C

*

*

**

*

*

*

*

*


at ASPE

T Journals on N

ovember 20, 2020

molpharm

.aspetjournals.orgD

ownloaded from


camp dependent protein kinase (pka)-mediated c-myc...

Documents