Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1478-1479
CORRESPONDENCE
Relationship Between Degradation of PML-RAR
and
Differentiation
 |
LETTER |
To The Editor:
In a recent issue of Blood, Fanelli et al1 reported
that constitutive degradation of PML-RAR
through the proteasome
pathway mediates retinoic acid (RA)-resistance in a derivative cell
line of NB4 (NB4.007/6). The context of this study contradicts our hypothesis that the degradation of PML-RAR is important as a trigger
of RA-dependent differentiation,2,3 although the conclusion
is reasonable that PML-RAR expression is crucial to RA-sensitivity.
To resolve the complexity of the relationship between the degradation
of PML-RAR and differentiation, we hope to elucidate the following.
Firstly, it must be determined whether the sequences of
PML-RAR and RAR genes
are normal or mutated in NB4.007/6. In some RA-resistant APL cell
lines, mutations of the PML-RAR gene have been
reported in the AF-2 domain, which is important to
ligand-binding.3-5 Because a missense mutation and a
premature termination potentially cause instability of the protein, the
constitutive degradation of PML-RAR might be due to the mutation.
Secondly, the investigators did not present the immunostaining data of
endogenous PML or PML-RAR in NB4.007/6. Because PML-RAR was
quickly degraded, the immunostaining pattern might resemble the wild
pattern. However, the investigators stained exogenous PML-RAR
carrying green fluorescent protein (GFP), which showed a microgranular
pattern. This data showed the short life of PML-RAR but not the
subcellular localization.
Finally, our recent data suggested that constitutive degradation of
PML-RAR is not necessarily associated with RA-resistance. We
established a new subline of NB4 (NB4/As) that showed resistance to
As2O3: continuous growth without apoptosis and
differentiation in the medium containing 1 µmol/L
As2O3. In immunoblot analysis, a PML-RAR
band of this cell line disappeared in the presence with As2O3 and reappeared after the
removal of As2O3 (data not shown).
As2O3 is known to modify PML as well as to
accelerate the degradation of PML-RAR.6-8 Although the
molecular mechanism of the As-resistance is under investigation, the
addition of RA differentiated NB4/As and increased the nitroblue
tetrazolium (NBT) reduction activity even in the coculture with
As2O3 (Fig 1).
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| Fig 1.
Morphology of NB4 (A, B, and C) and NB4/As (D, E, and F)
cell lines on RA or the combination of RA and
As2O3 treatment. (A and D) Before treatment. (B
and E) After 1 day of treatment with 1 µmol/L
As2O3. (C) After 1 day of treatment with 1 µmol/L all-trans RA and 1 µmol/L
As2O3. (F) After 4 days of treatment with 1 µmol/L RA and 1 µmol/L As2O3.
|
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We considered that the pathophysiology of NB4.007/6 might have changed
from the parental cell line after culture with RA. Additional molecular
changes increase the complexity of the RA-resistance in NB4.007/6.
Investigation of the RA-resistance cell lines has shown that PML-RAR
is a direct target molecule for RA. However, the relationship between
the degradation of PML-RAR and differentiation remains to be
resolved.
Tomoki Naoe
Kunio Kitamura
Department of
Infectious Diseases
Nagoya University School of Medicine
Nagoya,
Japan
| |
REFERENCES |
1.
Fanelli M, Minucci S, Gelmetti V, Nervi C, Gambacorti-Passerini C, Pelicci PG:
Constitutive degradation of PML/RAR through the proteasome pathway mediates retinoic acid resistance.
Blood
93:1477, 1999[Abstract/Free Full Text]
2.
Yoshida H, Kitamura K, Tanaka K, Omura S, Miyazaki T, Hachiya T, Ohno R, Naoe T:
Accelerated degradation of PML-retinoic acid receptor alpha (PML-RARA) oncoprotein by all-trans-retinoic acid in acute promyelocytic leukemia: Possible role of the proteasome pathway.
Cancer Res
56:2945, 1996[Abstract/Free Full Text]
3.
Kitamura K, Kiyoi H, Yoshida H, Saito H, Ohno R, Naoe T:
Mutant AF-2 domain of PML-RAR in retinoic acid-resistant NB4 cells: Differentiation induced by RA is triggered directly through PML-RARalpha and its down-regulation in acute promyelocytic leukemia.
Leukemia
11:1950, 1997[Medline]
[Order article via Infotrieve]
4.
Shao W, Benedetti L, Lamph WW, Nervi C, Miller W Jr.:
A retinoid-resistant acute promyelocytic leukemia subclone expresses a dominant negative PML-RAR mutation.
Blood
89:4282, 1997[Abstract/Free Full Text]
5.
Nason-Burchenal K, Allopenna J, Begue A, Stehelin D, Dmitrovsky E, Martin P:
Targeting of PML/RAR is lethal to retinoic acid-resistant promyelocytic leukemia cells.
Blood
92:1758, 1998[Abstract/Free Full Text]
6.
Muller S, Matunis MJ, Dejean A:
Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus.
EMBO J
17:61, 1998[Medline]
[Order article via Infotrieve]
7.
Shao W, Fanelli M, Ferrara FF, Riccioni R, Rosenauer A, Davison K, Lamph WW, Waxman S, Pelicci PG, Lo Coco F, Avvisati G, Testa U, Peschle C, Gambacorti-Passerini C, Nervi C, Miller W Jr:
Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR protein in acute promyelocytic leukemia cells.
J Natl Cancer Inst
90:124, 1998[Abstract/Free Full Text]
8.
Gianni M, Koken MH, Chelbi-Alix MK, Benoit G, Lanotte M, Chen Z, de The H:
Combined arsenic and retinoic acid treatment enhances differentiation and apoptosis in arsenic-resistant NB4 cells.
Blood
91:4300, 1998[Abstract/Free Full Text]
Response
One of the conclusions of our report1 is that
PML/RAR itself mediates RA-dependent differentiation and that its
degradation by RA is not crucial for the differentiation process to
occur. Naoe and Kitamura disagree.
In general, we think that there is much direct and indirect evidence to
support these conclusions: (1) PML/RAR is an RA-dependent transcriptional activator2,3; (2) PML/RAR mediates
RA-induced differentiation in both APL and non-APL cells; in
particular, PML/RAR restores RA-sensitivity in RA-resistant cell
lines carrying mutations of RAR4,5; (3) commitment to
differentiation in the presence of RA occurs within the first 12 hours,
when PML/RAR is still detectable6; (4) inhibition of
PML/RAR degradation by caspase inhibitors (another mediator of
RA-induced degradation of the fusion protein) increases RA-induced
differentiation in APL cells7; and (5) PML/RAR
re-expression restores RA-sensitivity in RA-resistant APL cells
with constitutive degradation of the fusion protein.1
With respect to some specific criticisms of Naoe and Kitamura: (1) we
did not sequence RAR and PML/RAR transcripts in NB4.007/6 cells.
Therefore, as suggested, we cannot exclude that mutations of the
PML/RAR coding sequence might affect fusion protein stability. However, we think that other mechanisms are more likely, because exogenous PML/RAR (GFP-PML/RAR) or PML/RAR from parental NB4 cells were equally degraded in NB4.007/6 cells in infection and in
vitro degradation assay, respectively. (2) Immunostaining of NB4.007/6
cells showed a micropunctuated pattern, typical of PML/RAR expression. Because we did not detect PML/RAR by Western blotting, this might reflect a better efficiency of our PG-M3 anti-PML monoclonal antibody on undenatured, versus denatured, antigens.
Finally, Naoe and Kitamura mentioned the isolation of an
arsenic-resistant/RA-sensitive NB4 subline. Because the signaling pathway triggered by arsenic is yet undefined, we do not think that
this model system can provide information as to the role of
PML/RAR in RA-signaling.
Mirco Fanelli
Pier
Giuseppe Pelicci
Department of Experimental Oncology
European
Institute of Oncology, Milan, Italy
| |
REFERENCES |
1.
Fanelli M, Minucci S, Gelmetti V, Nervi C, Gambacorti-Passerini C, Pelicci PG:
Constitutive degradation of PML/RAR through the proteasome pathway mediates retinoic acid resistance.
Blood
93:1477, 1999
2.
Grignani Fr, De Matteis S, Nervi C, Tomassoni L, Gelmetti V, Cioce M, Fanell M, Ruthard M, Ferrara FF, Zamir I, Seiser C, Grignani F, Lazar MA, Minucci S, Pelicci PG:
Fusion proteins of retinoic acid receptor- recruit histone deacetylase in promyelocytic leukaemia.
Nature
391:815, 1998[Medline]
[Order article via Infotrieve]
3.
Lin RJ, Nagy L, Inoue S, Shao W, Miller Jr WH, Evans RM:
Role of the histone deacetylase complex in acute promyelocytic leukaemia.
Nature
391:811, 1998[Medline]
[Order article via Infotrieve]
4.
Grignani Fr, Ferruci PF, Testa U, Talamo G, Fagioli M, Alcalay M, Mencarelli A, Grignani F, Peschle C, Nicoletti I, Pelicci PG:
The acute promyelocytic leukemia-specific PML/RAR fusion protein inhibits differentiation and promotes survival of myeloid precursor cells.
Cell
74:423, 1993[Medline]
[Order article via Infotrieve]
5.
Ruthardt M, Testa U, Nervi C, Ferruci PF, Grignani F, Puccetti E, Grignani F, Peschle C, Pelicci PG:
Opposite effects of the acute promyelocytic leukemia PML-retinoic acid receptor alpha (RAR alpha) and PLZF-RAR alpha fusion proteins on retinoic acid signalling.
Mol Cell Biol
17:4859, 1997[Abstract]
6.
Casini T, Pelicci PG:
A function of p21 during promyelocytic leukemia cell differentiation independent of CDK inhibition and cell cycle arrest.
Oncogene
18:3235, 1999[Medline]
[Order article via Infotrieve]
7.
Nervi C, Ferrara FF, Fanelli M, Rippo MR, Tomassini B, Ferrucci PF, Ruthardt M, Gelmetti V, Gambacorti-Passerini C, Diverio D, Grignani F, Pelicci PG, Testi R:
Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RAR fusion protein.
Blood
92:2244, 1998[Abstract/Free Full Text]
