(Received for publication, August 18, 1995; and in revised form, October 30, 1995)
From the
The promyelocytic leukemia gene (PML) involved in the t(15;17) (q22;q12) translocation in acute promyelocytic leukemia is a growth suppressor. To elucidate the functional domains of PML, several mutants lacking the nuclear localization signal (PMLnls-), the dimerization domain (PMLdim-), the proline-rich domain at the N-terminal (PMLpro-), the proline-rich RING finger motif (PMLpr-), the proline-rich RING finger B-box-1 (PMLprb-), the serine-proline-rich domain at the C-terminal (PMLsp-), and the double mutant (PMLprb-nls-) have been constructed. Immunofluorescence staining of transiently transfected NIH3T3 cells demonstrated that the RING finger motif, dimerization domain, and nuclear localization signal are all required for the formation of PML oncogenic domains (PODs). Immunofluorescence staining of transiently transfected GM637D human fibroblasts indicated that expression of PMLprb-, PMLnls-, and PMLprb-nls- led to a significant reduction or, in some cases, complete elimination of PODs. PMLdim-, PMLnls-, PMLpr-, PMLprb-, and PMLprb-nls- mutants were found to lose their ability to suppress transformation of NIH3T3 cells by activated neu, while PMLpro- and PMLsp- mutants did not. These results suggest that the ability of PML to form a POD is essential for suppression of growth and transformation. Furthermore, since PMLprb-, PMLnls-, and PMLprb-nls- mutants could block the suppression effect of wild-type PML on transformation of NIH3T3 cells by the neu oncogene, these PML mutants are potential dominant negative inhibitors of PML. Our study also suggests that the RING finger motif may interact with other nuclear proteins.
The nonrandom chromosomal translocation t(15;17) is a
cytogenetic hallmark of acute promyelocytic leukemia
(APL)()(1) , and the genes involved at the
breakpoint site have been cloned and characterized (2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
It is now clear that the t(15;17) translocation fuses the retinoic acid
receptor-
(RAR
) gene on chromosome 17 and the PML gene on chromosome 15. Fusion genes PML-RAR
and RAR
-PML transcribe fusion transcripts and encode
potentially oncogenic fusion proteins. Since most of the functional
domains of both PML and RAR
are retained in the fusion protein
PML-RAR
, it is speculated that PML-RAR
is critical for the
pathogenesis of APL. Indeed, in cotransfection assays, it was found
that PML-RAR
represses RA-responsive promoters in the absence of
RA(5, 6, 7, 9) . The stable
expression of PML-RAR
inhibits apoptosis in U937 cells and makes
these cells unable to respond to differentiation induction by
1,25-dihydroxyvitamin D
(vitamin D
) and
transforming growth factor
(12) . In addition,
stable expression of PML-RAR
in K562 cells interferes with
erythroid differentiation induced by hemin(13, 14) .
Thus, expression of this fusion protein in APL cells may be responsible
for blocking differentiation and prolonging survival of promyelocytes.
PML-RAR can effectively sequester PML and the retinoic X
receptor (RXR)(7, 15, 16, 17) . In
APL cells, double-color immunofluorescence staining of PML, RXR, and
PML-RAR
demonstrated that both PML and RXR colocalize with
PML-RAR
in vivo(16) . However, after
all-trans-retinoic acid (ATRA) induced differentiation of the
APL cells, PML and RXR no longer colocalized with PML-RAR
,
indicating that sequestration of PML and RXR by the PML-RAR
fusion
protein is critical for the development of APL. This notion is strongly
supported by our recent finding that PML is a growth
suppressor(18) . In brief, we found that (i) increased
expression of PML in the APL cells results in a loss of clonogenicity
in soft agar assay and tumorigenicity in nude mice(18) ; (ii)
PML suppresses the transformation of rat embryo fibroblasts by
cooperative oncogenes and of NIH3T3 cells by activated neu(18, 19) ; (iii) PML is a
promoter-specific transcription suppressor, suppressing the promoter
activity of the epidermal growth factor receptor and
multidrug-resistance genes and having little effect on the promoters of
-actin, SV-40, and Rous sarcoma virus(18) . From this
study and others, we hypothesized that PML-RAR
plays a central
role in APL as a dominant negative inhibitor of the normal function of
PML (growth suppressor) and RXR (coregulator of RAR, thyroid hormone,
vitamin D receptor, etc.) and consequently leads to the development of
APL by growth stimulation and loss of differentiation induction by
hormone or growth factors.
PML is a member of the family of RING finger proteins capable of forming a homodimer through the dimerization domain(20) . As shown by immunofluorescence staining, PML accumulates in the nucleus in a speckled pattern composed of nuclear bodies called PML oncogenic domains (PODs)(17) . PODs are also known as ND10 (21) or Kr bodies (16) . PML is believed to colocalize with the autoantigens in primary biliary cirrhosis, but the two proteins are not directly known to interact with each other(16, 17, 22) . It is speculated that PML first interacts with other unknown protein factors and then is indirectly associated with the autoantigens. This type of PML-associated protein factor has yet to be identified. In APL cells induced to differentiated by ATRA, the abnormal microspeckled PML pattern can be reorganized to the normal PODs(16, 17) . In RA-resistant, APL-derived NB4 cells, POD formation cannot be induced by ATRA treatment(17) . Together, these results indicate that the ability of PML to form PODs is required for the induction of differentiation in APL cells.
Taking advantage of the fact that PML can suppress the formation of NIH3T3 cell foci by activated neu(18, 19) , we have constructed several PML mutants and examined their ability to form PODs in vivo and suppress transformation. Here, we demonstrate that PML mutants lacking the nuclear localization signal, the dimerization domain, and the RING finger domain are unable to form PODs and that their ability to suppress foci formation is significantly reduced. However, mutants lacking the proline-rich domain at the N-terminal and the serine-proline-rich domain on the C-terminal can form PODs in vivo and can suppress foci formation, much like the wild-type PML. Our results corroborate the previous finding that the ability of APL cells to reassemble PODs after ATRA treatment is an important event in differentiation induction(17) . Furthermore, we show that PMLprb-, PMLnls-, and PMLprb-nls- are potential dominant negative inhibitors of PML and that the RING finger motif may interact with other nuclear proteins.
Figure 1: Schematic showing the structural features of wild-type PML protein and its mutants.
Briefly, exponentially growing cells were trypsinized, seeded at 5
10
cells per 10-cm plate, and incubated overnight
in 10 ml of complete Dulbecco's modified Eagle's medium.
Then, 10-20 µg of plasmid DNA premixed with 0.5 ml of 0.25 M CaCl
, 0.5 ml of 2
Hanks' buffered
solution (50 mM Hepes, 280 mM NaCl, 1.5 mM
Na
HPO
, pH 7.04 ± 0.02) was added, and
the mixture was incubated for 15-20 min at room temperature.
Calcium phosphate-DNA solution (1 ml) was added dropwise to the plate
of cells, after which the mixture was swirled gently and incubated for
16-24 h at 37 °C under 5% CO
. The medium was
removed, and the cells were rinsed twice with growth medium, refed, and
incubated for 24 h at 37 °C under 5% CO
. The cells were
split at a 1:10 ratio for focus-forming assay.
To confirm that these mutant clones would translate their respective mutant PML proteins, each of these plasmids were transfected into NIH3T3 cells by lipofectamine-mediated gene transfer. Western blot analysis of protein fractions isolated from these cells indicated that PMLdim- and PMLnls- encoded mutant PML proteins of the expected size (approximately 80-90 kDa) (Fig. 2A, lanes 6 and 7). However, mutant PMLpro-, PMLpr-, and PMLprb- encoded mutant proteins significantly smaller than expected. We have previously reported that although its predicted size is about 72 kDa, the PML protein migrates at about 90 kDa during SDS-polyacrylamide gel electrophoresis, probably due to its acidic nature(18) . As shown in Fig. 2A, lane 2, PMLpro- with a deletion of the first 48 amino acids migrated at about 70 kDa, indicating that is much smaller than the wild-type PML. To confirm that there is no additional deletion within the cDNA sequence, we completely sequenced one strand of PMLpro- and found no deletion in any other region of the cDNA. We speculate that the significant difference in size between PMLpro- and PML is the result of a change in secondary structure. The proline-rich domain may be responsible for the slower running nature of the wild-type PML in SDS-polyacrylamide gel electrophoresis. This notion is supported by the fact that both PMLpr- and PMLprb- migrated at molecular weights agreeable with their predicted sizes (Fig. 2A, lanes 3-5).
Figure 2: Expression of mutant PML proteins in NIH3T3 cells. The NIH3T3 cells were transfected with the various PML mutant plasmids by lipofectamine (Life Technologies, Inc.). Total protein was isolated after 48 h of culture, and Western blotting was performed as described previously(18) . A, lane 1, wild-type PML; lane 2, PMLpro-; lane 3, PMLpr-; lanes 4 and 5, PMLprb-; lane 6, PMLdim-; lane 7, PMLnls-. B, in vitro transcription and translation of the PML mutant plasmids. In vitro transcription and translation were performed as described previously(18) . Lane 1, wild-type PML; lane 2, PMLpro-; lane 3, PMLpr-; lane 4, PMLprb-; lane 5, pSG5 (an empty vector used as a negative control). Protein size markers are indicated on the left of both panels in kilodaltons.
To further confirm these findings, the PML mutant plasmids were transcribed and translated in vitro. Fig. 2B showed that the molecular weights of the in vitro transcribed and translated PMLpro-, PMLpr-, PMLprb-, and PMLdim- proteins agree with those derived from the Western blotting analysis as shown in Fig. 2A.
Figure 3:
Cellular distribution of PML and its
mutants in transfected NIH3T3 cells. NIH3T3 cells transfected with
2-3 µg of the respective plasmids by lipofectamine were
cultured for 48 h, and immunofluorescence staining was performed as
described previously(19) . a, negative control; b, PMLnls-; c, PMLdim-; d,
PMLpro-; e, PMLpr-; f, PMLprb-; g, double mutant (PMLprb-nls-); h,
PMLsp-. Magnification, 1000.
Results of these studies (Fig. 4) showed that PMLdim-, PMLnls-, PMLpr-, PMLprb-, and PMLprb-nls- lost their ability to suppress transformation of NIH3T3 by activated neu. This suggests that the dimerization domain, nuclear localization signal, and RING finger motif are all required for the PML growth suppressor function. As shown in Fig. 3, all of these PML mutants share one common property, i.e. they are unable to assemble into a nuclear speckled pattern or the PODs. This suggests that the ability of PMLs to organize into PODs is required for PML to exert its growth or transformation suppressor function. This notion is supported by the finding that PML mutants PMLpro- and PMLsp-, which are capable of forming PODs (Fig. 3, a and d), were both able to suppress neu-induced transformation of NIH3T3 cells, just like the wild-type PML (Fig. 4).
Figure 4:
The effect of mutant PMLs on suppression
of neu-induced transformation of NIH3T3 cells. Cotransfection
of plasmids by calcium phosphate coprecipitation and focus-forming
assays were performed as described previously(18) . In each
transfection assay, 10 µg of plasmid was transfected. The results
shown represent means of three independent experiments with three
plates each. In each transfection, 2 µg of pSV-Gal
(
-galactosidase cDNA under the control of the SV40-early promoter)
was included, and the activity of
-galactosidase was determined to
monitor transfection efficiency. Plasmid c-neu104 containing
the activated neu oncogene has been described
previously(18) ; PML and its mutants used in the study
are described in Fig. 1.
Figure 5:
Cellular localization patterns of PML in
human fibroblast GM637D cells. The GM637D cells were transfected with
2-3 µg of the PML mutant plasmid constructs by
lipofectamine (Life Technologies, Inc.). After 48 h of culture,
immunofluorescence staining was performed as described
previously(19) . a, wild-type PML; b,
PMLprb-; c, double mutant (PMLprb-nls-); d, PMLnls-. Magnification,
1000.
The dominant negative
inhibitory effect of PMLprb-, PMLnls-, and
PMLprb-nls- were further demonstrated by their ability to
inhibit the biologic function of the wild-type PML. As shown in Fig. 6, PMLprb-, PMLnls-, and
PMLprb-nls- were remarkably capable of knocking out the
ability of wild-type PML to suppress transformaton of NIH3T3 cells by
activated neu. This inhibition is similar to that caused by
the PML-RAR fusion protein encoded from the breakpoint of the
t(15;17) translocation in APL(18) . Furthermore, PMLprb-
and PMLprb-nls- inhibited PML function in a dose-dependent
manner (data not shown).
Figure 6:
PMLprb-, PMLnls-, and
PMLprb-nls- as potential dominant negative inhibitors of
the wild-type PML. Cotransfection of plasmids by calcium phosphate
coprecipitation and focus-forming assays were performed as described
previously(18) . In each transfection assay, 10 µg (unless
otherwise specified in parentheses) of plasmid was transfected. The
results shown represent the means of three independent experiments with
three plates each. Plasmid pSV-Gal was included, and the activity
of
-galactosidase was determined to monitor transfection
efficiency.
To understand the function of PML, a growth suppressor involved in the t(15;17) translocation in APL, we constructed several mutant plasmids and used them to analyze its functional domains. We found that (i) the nuclear localization signal, dimerization domain, and RING finger motif of PML are all essential for the formation of PODs; (ii) the ability of PML to form PODs in vivo is required for its growth suppressor function; and (iii) PMLprb-, PMLnls-, and PMLprb-nls- are potential candidates for dominant negative inhibition of the wild-type PML. Moreover, we have uncovered evidence that the RING finger motif of PML may interact with other proteins.
The PML protein contains several important domains,
including a C3HC4 (3 Cys, 1 His, and 4 Cys) zinc-finger motif (RING
finger domain); two additional Cys/His regions, referred to as B-box-1
and B-box-2; and an -helical coiled-coil
region(21, 26, 27, 28) .
Immunofluorescence staining demonstrated that PML is a major component
of novel nuclear bodies designated
PODs(17, 20, 26) . We have conclusively
demonstrated that the nuclear localization signal, the dimerization
domain, and the RING finger motif of PML, but not the proline-rich
domain at the N-terminal and the serine-proline-rich domain at the
C-terminal, are required for the formation of PODs. Our results, as
shown in Fig. 3, e and f, agree with the
observation of Kastner et al.(7) that PML proteins
bearing a mutation in the cysteine-rich (RING finger) motif
(Glu
-Cys
Glu
-Leu
) had a diffuse nuclear staining
pattern. During the preparation of this manuscript, Borden et al.(29) also demonstrated by site-directed mutagenesis that
the RING finger motif of PML is necessary for POD formation.
In our previous reports, we showed that PML is a growth and transformation suppressor(18, 19) . In this study, we demonstrated that the RING finger motif, dimerization domain, and nuclear localization signal are responsible for this suppression of growth and transformation. Since these regions of PML are also required for the formation of PODs, we therefore concluded that the ability of PML to assemble into PODs is essential for the growth suppression effect of PML. This finding is corroborated by the observations that ATRA-induced differentiation of NB4 and APL cells is associated with a reorganization of the microspeckled nuclear pattern into PODs and that no such response was found in the ATRA-resistant NB4 cells(16, 17, 22) .
At present, the composition and function of PODs is not completely understood. Our finding that PML is a growth and transformation suppressor sheds new light on the in vivo biologic function of this new form of nuclear body. PODs, which are associated with the nuclear matrix, reportedly do not contain any of the proteins known to be involved in pre-mRNA splicing, transcription, and DNA synthesis (17, 30) . In addition to PML, the PODs may include the SP100, NDP-55, and Vmw110 (also known as ICP0) proteins and an unidentified 65-kDa protein(16, 17, 21, 22, 30) . These proteins, however, do not directly interact with PML. In studies of APL and herpes simplex virus immediate-early protein, PODs appeared to be extremely important (both directly and indirectly) for myeloid differentiation as well as regulation of gene expression (e.g. activation of both cellular and viral genes in infection)(16, 17, 21, 22, 30, 31) .
Recently, the growth suppressor function of PML was confirmed by another group, who found that when skin, breast, and colon malignant cells turned invasive, PML expression was lost(32) . These data imply that PML may be an antioncogene involved not only in APL but also in non-hematological oncogenesis. Interestingly, Terris et al.(33) reported that expression of PML increased significantly in the inflammatory tissues and during normal or pathological proliferative states, indicating that PML plays a role in the inflammatory process and in cell growth control.
As stated
above, the PML-RAR fusion protein is reportedly a dominant
negative inhibitor of PML and
RXR(7, 17, 34) . Previous immunofluorescence
staining of PML indicated cytoplasmic and/or nuclear localization with
a micropunctuated pattern in APL blasts and in cells stably transfected
with PML-RAR
. Here, we have demonstrated that PMLprb-,
PMLnls-, and PMLprb-nls- are potential dominant
negative inhibitors against the normal function of PML and that
transient expression of PMLprb-, PMLnls-, and
PMLprb-nls- significantly reduces or completely eliminates
normal PODs in GM637D cells. Furthermore, we have shown that
PMLprb-, PMLnls-, and PMLprb-nls- can block the
effect of wild-type PML on transformation of NIH3T3 cells by activated neu. We have noticed that stable transfectants overexpressing
PMLnls- in GM637D cells eventually died (data not shown). Since
overexpression of PML in B104-1-1 (19) and HeLa cells (
)did not result in cell death, we therefore reason that
PMLnls- probably induced the observed cell death through its
perinuclear localization. It is possible that the RING finger motif
interacts with other important unknown proteins essential for cell
growth. Sequestration of these proteins by PMLnls- may be
responsible for the cell death. Therefore, PMLnls- may not be a
suitable dominant negative inhibitor. Conversely, PMLprb- and
PMLprb-nls-, both without the RING finger motif, should be
more suitable for testing the effect of knocking out wild-type PML in
normal cells and for studying whether elimination of cellular PML will
result in growth stimulation. We plan to establish in our laboratory
stable transfectants of GM637D constitutively expressing the
PMLprb- and PMLprb-nls- and to investigate the
effects of these dominant negative inhibitors on proliferation and
growth of GM637D cells. Such studies will be helpful in further
understanding the function of PML in the control of cell growth.
As
stated above, PML is characterized by the RING finger motif. This motif
has the sequence
C-X
-C
-X
-C
-X
-H
-X
-C
-X
-C
-X
-C
-X
-C
,
where C represents cysteine, H represents histidine, X can be
any amino acid, and the numbers in parentheses refer to the length in
amino acids of the sequences(20, 27) . The RING finger
motif may play a important cellular role by acting as a DNA binding or
protein-protein interacting
domain(9, 20, 29) . It is necessary then to
determine whether DNA or protein is involved in the interaction with
the RING finger motif of PML protein. The fact that treatment of the
nuclei with RNase and DNase in previous studies did not disrupt the
PODs in human cell lines suggests that nucleic acids are not a
component of this structure (30) . (
)Consequently,
this implies that it is the RING finger motif that normally interacts
with protein factors. Our results presented in Fig. 3and Fig. 4show that the immunostaining pattern of mutant
PMLnls- is clearly distinct from that of double mutant
PMLprb-nls- and that PMLnls- accumulated in the
cytoplasm, mostly in the perinucleus. In contrast, the staining pattern
of the double mutant was fine and diffuse throughout the cytoplasm.
This suggests that the RING finger motif accounts for these
differences. We therefore speculate that the RING finger motif of PML
interacts with other nuclear protein factors and is responsible for the
differences in cellular distribution between PMLnls- and
PMLprb-nls-.
We conclude then that the RING finger motif,
-helical region, and nuclear localization signal of PML are all
required for the formation of PODs. We also conclude that the ability
of PML to form PODs is essential for its growth and transformation
suppressor function. However, studies are needed (i) to define more
precisely the specific contribution of each domain in PML and (ii) to
define the composition and function of PODs so as to understand in
depth their role during human oncogenesis. In this direction, we have
been assessing the effects of the two dominant negative inhibitors of
wild-type PML described in this report (PMLprb- and
PMLprb-nls-) on the proliferation and differentiation of
GM637D cells, which stably express mutant PMLs.