AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 780-787
BIOCHEMICAL
Vol. 169, No. 2, 1990 June 15, 1990
POST-TRANSCRIPTIONAL REGULATION OF THYMIDINE KINASE GENE EXPRESSION DURING MONOCYTIC DIFFERENTIATION OF HL60 PROMYELOCYTES Zee-Fen Chang Department
Received
of Biochemistry, Chang Gung Medical College, Tao-Yuan, Taiwan, R.O.C.
May 7, 1990
SUMMARY : The regulatory mechanism of human thymidine kinase (TK) gene expression was investigated in HL-60 promyelocytes during induction of monocytic differentiation with 12-0-tetradecanoyl phorbol-13-acetate (TPA). The steady-state levels of TK mRNA diminished gradually as cells were treated with TPA. The nuclear run-on experiments were pet-formed and revealed that TPA treatment did not change TK gene activity in HL-60 cells. These findings suggested that the expression of TK mRNA was controlled by a post-transcriptional mechanism. The half-life of mature TK mRNA transcript was found to be more than 8 hours in both proliferating and differentiated HL-60 cells, which indicated that the stability of mature TK mRNA does not play a role in regulating TK gene expression. Analysis of poly(A-) TK mRNAs showed the high molecular weight precursors of TK mRNA which appeared in proliferating cells were not detectable in TPA-treated cells. This finding suggested that the TK mRNA processing event is implicated in the regulation of human TK gene expression in HL-60 cells during monocytic terminal differentiation. Q1990 kademic Press, Inc. Thymidine
kinase (TK) is an enzyme in the pyrimidine
that catalyzes the phosphorylation synthesis of TK is interesting
of thymidine
to dTMP.
salvage pathway
The regulation
of the
because its activity rises as cells enter S phase
of the cell-cycle and declines as cells withdraw from the cell cycles [I ,2,3]. The TK gene provides a useful model system for understanding
the molecular
basis of genes that are closely associated
Studies on
the serum stimulation
with DNA replication.
and simian virus infection of growth-arrested
cells have shown that TK is mainly controlled post-transcriptional mRNA in embryonic 0006-291X/90 Copyright All rights
levels [4]. Groudine
and Casimir have indicated
chicken cells was primarily
$1.50
0 I990 by Academic Press, Inc. of reproduction in any form reserved.
780
at both transcriptional
regulated
by a
CV-1 and that TK
Vol. 169, No. 2, 1990
post-transcriptional demonstrated
mechanism
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
[5]. Contrary to this result,
that upon terminal
is transcriptionally prompted
BIOCHEMICAL
differentiation
it has been
in mouse myoblasts
repressed [6]. The controversy
TK gene
among these studies
us to use other system to study the regulatory
mechanism
of TK gene
expression. The present investigation TK gene expression
used HL-60 cells to examine
during its monocytic
terminal
the mechanism
differentiation
71. Cultured HL-60 cells could be induced to undergo terminal into adherent cessation
macrophages
system to study the regulation cellular terminal
that the great decrement
The steady-state
not in TPA-treated
during
level of TK mRNA was
at 48 hours of treatment.
The results demonstrated
of TK mRNA level during the monocytic
of HL-60 cells is neither due to repression
weight
with
it provides a valuable
kinase gene expression
nor to the change in the stability of mature TK mRNA. molecular
differentiation
as HL-60 cells were treated with TPA for 24 hours and
was further diminished
differentiation
of thymidine
differentiation.
shown to decrease
[reviewed in
by TPA and this process is also associated
of DNA synthesis [reviewed in 71. Therefore
of
of TK gene activity, The presence of high
TK transcript precursors in proliferating cells suggested
mainly during the processing
that TK gene expression
HL-60 cells but may be regulated
events of TK mRNA.
MATERIAL
AND METHODS
Chemical TPA and Actinomycin D were purchased from Sigma Chem. Co., St. Louis, MO. [cx-~*P] dCTP and [a-32P] UTP were from Amersham. All other chemicals were of standard reagent grade. Cell Culture HL-60 cells were obtained from American Type Culture Collection, and grown in RPMI-1640 (GIBCO) supplemented with 10% heat inactivated fetal bovine serum (GIBCO) in a humidified atmosphere of 95% air, 5% CO,. In all experiments, proliferating cells were inoculated at a concentration differentiation concentration
of 5 x 1 O5 cells/ml in fresh medium. For monocytic of l-IL-60 cells, the cells were incubated with TPA at a of 17 nM for the periods indicated.
RNA using a guanidium
. .
.
isothiocyanate
Total cellular RNA was prepared from cells procedure described by Chomczynski and 781
Vol.
169,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Sacchi [a]. Cell pellets were lysed and vigorously homogenized in a solution containing 4 M guanidium thiocyanate, 25 mM sodium citrate, 200mM sodium acetate, pH 4.0, 0.5% sarcosyl, 0.1 M j3-mercaptoethanol, total RNA was then phenol extracted and isopropyl precipitated. Polyadenylated poly(A+) RNA was prepared from total RNA by affinity chromatography on an oligo(dT) cellulose column [9]. Designated amount of RNA was analyzed by formaldehyde gel electrophoresis [I 01, transferred to nitrocellulose paper (Schleicher & Schuell), and hybridized to appropriate DNA probes by described methods [l 11. The pTK11 containing 1.4 Kb of human TK cDNA insert [12] and the 2.0 Kb of Pstl fragment of the chicken 8-actin cloned in pGEM 2 [13] were generous gifts from Dr. Prescott L. Deninger and Dr. L. N. Wei, respectively. Both DNA probes were labeled by primer extension with [o~-~~P] dCTP to yield specific activities of approximately 2x lo8 cpm/ug [14]. Filters were then washed , dried and exposed to Kodak XAR-5 film for 2 to 24 hours. Relative signal was determined by densitometric scanning (Hoefer scientific instrument). Nuclear Run-on Assavs Cells were treated as indicated, pelleted at lOOOxg, and washed twice with ice-cold phosphate-buffered saline. The cells were then suspended in 5 ml of ice-cold lysis buffered( 10mM Tris-HCI [pH 7.41, 1 OmM NaCI, 3 mM MgCI,, 0.5% Nonidet P-40 ), mixed gently and left on ice for 5 min. Nuclei were then purified and used for synthesizing the nuclear transcripts as described by Mitchell et al. [15] except the KCI concentration was 50 mM, since the high concentration of monovalent cation was found to cause HL-60 cells clumping; and r2P]UTP was included in the transcription buffer at 2.5 mCi/ ml. Nuclear labeled RNA was isolated in 0.25% SDS/ 1 OmM Tris/ 1 mM EDTA , pH7.1 with a centriconemicroconcentrator (Amicon). The pTK11 , pActin and pBR322 were denatured in 0.3 M NaOH for 30 min at 65OC. After neutralization, 10 ug of plasmid was blotted directly per well on nitrocellulose paper by using dot manifold. DNA dot blots were hybridized with 1 06cpm/ml for 48 hours and washed at 55OC in 0.2xSSC/O.l %SDS before autoradiography.
RESULTS
AND DISCUSSION
HL-60 cells are induced to differentiate
along the monocytic
lineage
following exposure to phorbol esters, such as TPA . In the present work, we found that the steady-state
level of TK mRNA in HL-60 cells
progressively
after treatment
disappeared
the level of TK mRNA was not changed by 40% (determined
by densitometric
at 48 hours with TPA treatment.
with TPA . As shown in Figure 1, at 8 hours of treatment,
scanning)
but decreased
at 24 hours and disappeared
Since analysis of total RNA by dot-blot
(data not shown) also indicated that total TK mRNA transcripts 782
were declined
Vol.
169, No. 2, 1990
Time (hr) 0
BIOCHEMICAL
8
24
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
48
Prolife
rating
18s. 1.5Kbd Actino mycin D 2 hr
+TPA 24 hr
+TPA
pactin
01
48
123
4
hr
02
Fiaure 1. Effects of TPA on TK mRNA levels. Proliferating HL-60 cells were treated with 17nM TPA for the indicated time. Poly(A+) RNA (5 pg) was analyzed by Northern blot as described previously and hybridized to 32P-labeledpTKl1. The same blot was later rehybridized with a 6-actin probe. Fiaure 2. Run-on transcription analysis. Nuclei were isolated from proliferating HL-60 cells and cells treated with 17 nM TPA (24 h, 48 h), or actinomycin D (5 Kg/ml, 2 h) and used for run-on assays performed as described in Methods and Materials The 32Plabeled nuclear RNA was hybridized to the indicated plasmid DNAs that had been spotted onto filters.
with TPA treatment,
it is, therefore,
process of polyadenylation
unlikely that the alteration
could be accounted
of the
for the change of mature TK
mRNA level. Run-on transcription of TK gene transcription actinomycin
D treated,
assays were performed
to determine
in nuclei isolated from proliferating 24 hours and 48 hours of TPA-treated 783
relative rates
, 2 hours of HL-60 cells.
Vol.
BIOCHEMICAL
169, No. 2, 1990
The hybridization
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of labeled nuclear RNA to pBR322
as the negative and positive control, respectively. the extremely
gene activity was not repressed and suggested
in these monocytic
differentiated
HL-60 cells
decrease in TK mRNA levels is due to a
mechanism.
In order to determine
Proliferating
in proliferating
(5 ug) and TPA-treated
D for the indicated
isolated and analyzed
by Northern
D exposure
in HL-60 cells. The declines
cytotoxicity
of this agent.
(48 hours)
(15 ug) cells were treated
time periods and poly (A+) RNA was blot, As shown in Figure 3, it seems that
The nuclear run-on assay indicated
TK RNA synthesis was 90% inhibited
D treatment
and TPA-treated
had very little effect on the mature TK and actin mRNA
during the initial 2 hour-treatment.
actinomycin
The
the stability of the TK mRNA transcript.
decay of TK mRNA was examined
with actinomycin
active in
This result revealed clearly that TK
that the TPA-induced
post-transcriptional
actinomycin
As seen in Figure 2, despite
low level of TK RNA, the TK gene was transcriptionally
cells with 48 hours of TPA treatment.
HL-60 cells,
and p-actin probe served
by two hours of actinomycin
of TK and p-actin mRNAs
in TPA-treated
that
D treatment
after 8 hours of
cells may be the result of
The result indicated that the half-life of mature
TK mRNA in HL-60 cells was more than 8 hours. This is similar to the data obtained
in mouse 3T3 cells [17], which showed that the
half-life of TK mRNA in S phase is around 8 to 12 hours.
The experiment
revealed that the stability of TK mRNA was not significantly TPA-treated
decreased
HL-60 cells. Thus, the stability is not responsible
decrease of steady-state
also
in
for the
level of TK mRNA during monocytic
differentiation
of
HL-60 cells. To investigate expression,
the possible role of RNA processing
the nonpolyadenylated
fraction of oligo(dT)
RNA was collected
affinity chromatography
As shown in
weight 7.5,4.1,
and 1.8 Kb
with TK cDNA probe and the 4.1 Kb appeared
major species of nonpolyadenylated
TK mRNA. 784
TK gene
from the unbound
and analyzed.
Figure 4, there were several bands at molecular which were hybridizable
in regulating
Presumably,
as the
these high
Vol.
BIOCHEMIC :ALAND BIOI ‘HYSICAL RESEARCH COMMUNICATIONS
169, No. 2, 1990
Ti
Proliferating Time(hr)
0
2
6
me
(hr)
0
24
48
TPA 4% hr 0
2
-4 !8S
8
-1 8s
1.8 +
04
456
Fiaure 3. Stability of TK mRNA. Actinomycin D (5 pg/ml) was added to the cultures of proliferating and TPA-treated (48 h) HL-60 cells. The total RNA was prepared from cells harvested at the times indicated. The Northern blot of the poly(A+) RNA was probed with labeled pTKl1 and 6-actin. Lanes : 1 through 3,5 Kg of RNA: 4 through 6, 15 pg of RNA. The blots probed by pTK11 were exposed to film for 6 hours. When probed by p-actin, the exposure time was 2 h for lane 1 to 3 and 30 min for lane 4 to 6 Fiqure 4. Effect of TPA on nonpolyadenylated forms of TK mRNA. Proliferating HL-60 cells were treated with 17nM TPA for the indicated time. Poly(A-) RNA (30 pg) was analyzed by Northern blot as described previously and hybridized to 32P-labeledpTKl1.
molecular
weight bands represented
heterogeneous
the processed
intermediates
of
It should be noted that 4.1 Kb of TK
nuclear (hn) TK mRNAs.
mRNA was not detected in cells treated
with TPA 24 hours, although
was 60% of mature TK mRNA remaining
in these cells. Hence, it is possible
that the newly synthesized
there
TK mRNA was not properly processed and become 785
BIOCHEMICAL
Vol. 169, No. 2, 1990
AND BIOPHYSICAL
the mature TK mRNA in the differentiated processing
RESEARCH COMMUNICATIONS
cells. The alteration
of this
event together with the result of the normal decay of TK mRNA may
explain the reduction It is interesting
of mature TK mRNA level in these differentiated
cells.
to note that Gudas et al. [17) have found that several nuclear
TK mRNA precursors with molecular
sizes at 8.8, 7.1, 6.4, 3.4 , 2.5 and 1.4 Kb
were present in the S phase of Balb/c 3T3 cells but were absent in quiescent cells.
They have suggested
that the lack of TK hnRNA processing
somehow signals the TK hnRNA degradation for the greatly diminished
in the nucleus, which may account
levels of mature TK RNA in quiescent
cells. Our results support their notion to some extent. findings suggested
the expression
HL-60 cells may be regulated
in G, cells
Balb/c 3T3
Taken together, these
of TK mRNA in monocytic-differentiated
by a post-transcriptional
control involved in
hnRNA splicing and turn over mechanisms.
ACKNOWLEDGMENTS This investigation was supported in part by grant CMRP262 from Chang Gung Medical College and a grant from NSC79-0412-Bl82-15 . I thank MS Duen-Yi Huang and Chiow-Jiau Lin for their able technical help and Dr. J.-K. Chen for his valuable advice. I am also grateful to Dr. Prescott L. Deninger and Dr. L. N. Wei for providing cDNA probes.
REFERENCES 1. Kit, S., Dubbs, D. R., and Frearson, P. M. (1965) J. Biol. Chem. 240, 2565-2573. 2. Johnson, L. F., Roa, L. G., and Muench, A. G. (1982) Exp. Cell Res. &j& 79-85. 3. Yang, H., and Pardee, A. B. (1986) J. Cell. Physiol. =,41 O-41 6. 4. Stewart, C. J., Ito, M., and Conrad, S. E. (1986) Mol. Cell. Biol. 4, 123-132. 5. Groudine, M., and Casimir, C. (1984) Nucleic Acids Res. 2, 1427-1446. 6. Gross, M. K., Kainz, M. S., and Merril, G. F. (1987) Dev. Biol. 122, 439-451. 7. Collin, S. J. (1987) Blood Zp, 1233-1244. 8. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. a, 156-l 59. 9. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408-1414. 10. Lehrach, H., Diamond, D., Wozney, J. M and Boedtker, H. (1977) Biochemistry =,4743-4751. 11. Thomas, P. (1980) Proc. Natl. Acad. Sci. USA a, 5201-5205. 12. Bradshaw, Jr., H.D., and Deininger, P. L. (1984) Mol. Cell. Biol. 4, 2316-2320. 786
Vol.
169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
13. Cleveland, D. W., Lopata, M. A., McDonald, R. J., Cowan, J. J., Flutter, W. J., and Kirschner, M. J. (1980) Cell 2p, 95-105 14. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. m, 6-13. 15. Mitchell, R. L., Henning-Chubb, C., Huberman, E., and Verma, J. M. (1985) Cell s, 495-504. 16. Coppock, D. L., and Pardee, A. B. (1987) Mol. Cell. Biol. Z, 2925-2932. 17. Gudas, J. M., Knight, G. B., and Pardee, A. B. (1988) Proc. Natl. Acad. Sci. USA a, 4705-4709.
787
BIOCHEMICAL
Vol. 169, No. 2, 1990 June 15, 1990
POST-TRANSCRIPTIONAL REGULATION OF THYMIDINE KINASE GENE EXPRESSION DURING MONOCYTIC DIFFERENTIATION OF HL60 PROMYELOCYTES Zee-Fen Chang Department
Received
of Biochemistry, Chang Gung Medical College, Tao-Yuan, Taiwan, R.O.C.
May 7, 1990
SUMMARY : The regulatory mechanism of human thymidine kinase (TK) gene expression was investigated in HL-60 promyelocytes during induction of monocytic differentiation with 12-0-tetradecanoyl phorbol-13-acetate (TPA). The steady-state levels of TK mRNA diminished gradually as cells were treated with TPA. The nuclear run-on experiments were pet-formed and revealed that TPA treatment did not change TK gene activity in HL-60 cells. These findings suggested that the expression of TK mRNA was controlled by a post-transcriptional mechanism. The half-life of mature TK mRNA transcript was found to be more than 8 hours in both proliferating and differentiated HL-60 cells, which indicated that the stability of mature TK mRNA does not play a role in regulating TK gene expression. Analysis of poly(A-) TK mRNAs showed the high molecular weight precursors of TK mRNA which appeared in proliferating cells were not detectable in TPA-treated cells. This finding suggested that the TK mRNA processing event is implicated in the regulation of human TK gene expression in HL-60 cells during monocytic terminal differentiation. Q1990 kademic Press, Inc. Thymidine
kinase (TK) is an enzyme in the pyrimidine
that catalyzes the phosphorylation synthesis of TK is interesting
of thymidine
to dTMP.
salvage pathway
The regulation
of the
because its activity rises as cells enter S phase
of the cell-cycle and declines as cells withdraw from the cell cycles [I ,2,3]. The TK gene provides a useful model system for understanding
the molecular
basis of genes that are closely associated
Studies on
the serum stimulation
with DNA replication.
and simian virus infection of growth-arrested
cells have shown that TK is mainly controlled post-transcriptional mRNA in embryonic 0006-291X/90 Copyright All rights
levels [4]. Groudine
and Casimir have indicated
chicken cells was primarily
$1.50
0 I990 by Academic Press, Inc. of reproduction in any form reserved.
780
at both transcriptional
regulated
by a
CV-1 and that TK
Vol. 169, No. 2, 1990
post-transcriptional demonstrated
mechanism
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
[5]. Contrary to this result,
that upon terminal
is transcriptionally prompted
BIOCHEMICAL
differentiation
it has been
in mouse myoblasts
repressed [6]. The controversy
TK gene
among these studies
us to use other system to study the regulatory
mechanism
of TK gene
expression. The present investigation TK gene expression
used HL-60 cells to examine
during its monocytic
terminal
the mechanism
differentiation
71. Cultured HL-60 cells could be induced to undergo terminal into adherent cessation
macrophages
system to study the regulation cellular terminal
that the great decrement
The steady-state
not in TPA-treated
during
level of TK mRNA was
at 48 hours of treatment.
The results demonstrated
of TK mRNA level during the monocytic
of HL-60 cells is neither due to repression
weight
with
it provides a valuable
kinase gene expression
nor to the change in the stability of mature TK mRNA. molecular
differentiation
as HL-60 cells were treated with TPA for 24 hours and
was further diminished
differentiation
of thymidine
differentiation.
shown to decrease
[reviewed in
by TPA and this process is also associated
of DNA synthesis [reviewed in 71. Therefore
of
of TK gene activity, The presence of high
TK transcript precursors in proliferating cells suggested
mainly during the processing
that TK gene expression
HL-60 cells but may be regulated
events of TK mRNA.
MATERIAL
AND METHODS
Chemical TPA and Actinomycin D were purchased from Sigma Chem. Co., St. Louis, MO. [cx-~*P] dCTP and [a-32P] UTP were from Amersham. All other chemicals were of standard reagent grade. Cell Culture HL-60 cells were obtained from American Type Culture Collection, and grown in RPMI-1640 (GIBCO) supplemented with 10% heat inactivated fetal bovine serum (GIBCO) in a humidified atmosphere of 95% air, 5% CO,. In all experiments, proliferating cells were inoculated at a concentration differentiation concentration
of 5 x 1 O5 cells/ml in fresh medium. For monocytic of l-IL-60 cells, the cells were incubated with TPA at a of 17 nM for the periods indicated.
RNA using a guanidium
. .
.
isothiocyanate
Total cellular RNA was prepared from cells procedure described by Chomczynski and 781
Vol.
169,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Sacchi [a]. Cell pellets were lysed and vigorously homogenized in a solution containing 4 M guanidium thiocyanate, 25 mM sodium citrate, 200mM sodium acetate, pH 4.0, 0.5% sarcosyl, 0.1 M j3-mercaptoethanol, total RNA was then phenol extracted and isopropyl precipitated. Polyadenylated poly(A+) RNA was prepared from total RNA by affinity chromatography on an oligo(dT) cellulose column [9]. Designated amount of RNA was analyzed by formaldehyde gel electrophoresis [I 01, transferred to nitrocellulose paper (Schleicher & Schuell), and hybridized to appropriate DNA probes by described methods [l 11. The pTK11 containing 1.4 Kb of human TK cDNA insert [12] and the 2.0 Kb of Pstl fragment of the chicken 8-actin cloned in pGEM 2 [13] were generous gifts from Dr. Prescott L. Deninger and Dr. L. N. Wei, respectively. Both DNA probes were labeled by primer extension with [o~-~~P] dCTP to yield specific activities of approximately 2x lo8 cpm/ug [14]. Filters were then washed , dried and exposed to Kodak XAR-5 film for 2 to 24 hours. Relative signal was determined by densitometric scanning (Hoefer scientific instrument). Nuclear Run-on Assavs Cells were treated as indicated, pelleted at lOOOxg, and washed twice with ice-cold phosphate-buffered saline. The cells were then suspended in 5 ml of ice-cold lysis buffered( 10mM Tris-HCI [pH 7.41, 1 OmM NaCI, 3 mM MgCI,, 0.5% Nonidet P-40 ), mixed gently and left on ice for 5 min. Nuclei were then purified and used for synthesizing the nuclear transcripts as described by Mitchell et al. [15] except the KCI concentration was 50 mM, since the high concentration of monovalent cation was found to cause HL-60 cells clumping; and r2P]UTP was included in the transcription buffer at 2.5 mCi/ ml. Nuclear labeled RNA was isolated in 0.25% SDS/ 1 OmM Tris/ 1 mM EDTA , pH7.1 with a centriconemicroconcentrator (Amicon). The pTK11 , pActin and pBR322 were denatured in 0.3 M NaOH for 30 min at 65OC. After neutralization, 10 ug of plasmid was blotted directly per well on nitrocellulose paper by using dot manifold. DNA dot blots were hybridized with 1 06cpm/ml for 48 hours and washed at 55OC in 0.2xSSC/O.l %SDS before autoradiography.
RESULTS
AND DISCUSSION
HL-60 cells are induced to differentiate
along the monocytic
lineage
following exposure to phorbol esters, such as TPA . In the present work, we found that the steady-state
level of TK mRNA in HL-60 cells
progressively
after treatment
disappeared
the level of TK mRNA was not changed by 40% (determined
by densitometric
at 48 hours with TPA treatment.
with TPA . As shown in Figure 1, at 8 hours of treatment,
scanning)
but decreased
at 24 hours and disappeared
Since analysis of total RNA by dot-blot
(data not shown) also indicated that total TK mRNA transcripts 782
were declined
Vol.
169, No. 2, 1990
Time (hr) 0
BIOCHEMICAL
8
24
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
48
Prolife
rating
18s. 1.5Kbd Actino mycin D 2 hr
+TPA 24 hr
+TPA
pactin
01
48
123
4
hr
02
Fiaure 1. Effects of TPA on TK mRNA levels. Proliferating HL-60 cells were treated with 17nM TPA for the indicated time. Poly(A+) RNA (5 pg) was analyzed by Northern blot as described previously and hybridized to 32P-labeledpTKl1. The same blot was later rehybridized with a 6-actin probe. Fiaure 2. Run-on transcription analysis. Nuclei were isolated from proliferating HL-60 cells and cells treated with 17 nM TPA (24 h, 48 h), or actinomycin D (5 Kg/ml, 2 h) and used for run-on assays performed as described in Methods and Materials The 32Plabeled nuclear RNA was hybridized to the indicated plasmid DNAs that had been spotted onto filters.
with TPA treatment,
it is, therefore,
process of polyadenylation
unlikely that the alteration
could be accounted
of the
for the change of mature TK
mRNA level. Run-on transcription of TK gene transcription actinomycin
D treated,
assays were performed
to determine
in nuclei isolated from proliferating 24 hours and 48 hours of TPA-treated 783
relative rates
, 2 hours of HL-60 cells.
Vol.
BIOCHEMICAL
169, No. 2, 1990
The hybridization
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of labeled nuclear RNA to pBR322
as the negative and positive control, respectively. the extremely
gene activity was not repressed and suggested
in these monocytic
differentiated
HL-60 cells
decrease in TK mRNA levels is due to a
mechanism.
In order to determine
Proliferating
in proliferating
(5 ug) and TPA-treated
D for the indicated
isolated and analyzed
by Northern
D exposure
in HL-60 cells. The declines
cytotoxicity
of this agent.
(48 hours)
(15 ug) cells were treated
time periods and poly (A+) RNA was blot, As shown in Figure 3, it seems that
The nuclear run-on assay indicated
TK RNA synthesis was 90% inhibited
D treatment
and TPA-treated
had very little effect on the mature TK and actin mRNA
during the initial 2 hour-treatment.
actinomycin
The
the stability of the TK mRNA transcript.
decay of TK mRNA was examined
with actinomycin
active in
This result revealed clearly that TK
that the TPA-induced
post-transcriptional
actinomycin
As seen in Figure 2, despite
low level of TK RNA, the TK gene was transcriptionally
cells with 48 hours of TPA treatment.
HL-60 cells,
and p-actin probe served
by two hours of actinomycin
of TK and p-actin mRNAs
in TPA-treated
that
D treatment
after 8 hours of
cells may be the result of
The result indicated that the half-life of mature
TK mRNA in HL-60 cells was more than 8 hours. This is similar to the data obtained
in mouse 3T3 cells [17], which showed that the
half-life of TK mRNA in S phase is around 8 to 12 hours.
The experiment
revealed that the stability of TK mRNA was not significantly TPA-treated
decreased
HL-60 cells. Thus, the stability is not responsible
decrease of steady-state
also
in
for the
level of TK mRNA during monocytic
differentiation
of
HL-60 cells. To investigate expression,
the possible role of RNA processing
the nonpolyadenylated
fraction of oligo(dT)
RNA was collected
affinity chromatography
As shown in
weight 7.5,4.1,
and 1.8 Kb
with TK cDNA probe and the 4.1 Kb appeared
major species of nonpolyadenylated
TK mRNA. 784
TK gene
from the unbound
and analyzed.
Figure 4, there were several bands at molecular which were hybridizable
in regulating
Presumably,
as the
these high
Vol.
BIOCHEMIC :ALAND BIOI ‘HYSICAL RESEARCH COMMUNICATIONS
169, No. 2, 1990
Ti
Proliferating Time(hr)
0
2
6
me
(hr)
0
24
48
TPA 4% hr 0
2
-4 !8S
8
-1 8s
1.8 +
04
456
Fiaure 3. Stability of TK mRNA. Actinomycin D (5 pg/ml) was added to the cultures of proliferating and TPA-treated (48 h) HL-60 cells. The total RNA was prepared from cells harvested at the times indicated. The Northern blot of the poly(A+) RNA was probed with labeled pTKl1 and 6-actin. Lanes : 1 through 3,5 Kg of RNA: 4 through 6, 15 pg of RNA. The blots probed by pTK11 were exposed to film for 6 hours. When probed by p-actin, the exposure time was 2 h for lane 1 to 3 and 30 min for lane 4 to 6 Fiqure 4. Effect of TPA on nonpolyadenylated forms of TK mRNA. Proliferating HL-60 cells were treated with 17nM TPA for the indicated time. Poly(A-) RNA (30 pg) was analyzed by Northern blot as described previously and hybridized to 32P-labeledpTKl1.
molecular
weight bands represented
heterogeneous
the processed
intermediates
of
It should be noted that 4.1 Kb of TK
nuclear (hn) TK mRNAs.
mRNA was not detected in cells treated
with TPA 24 hours, although
was 60% of mature TK mRNA remaining
in these cells. Hence, it is possible
that the newly synthesized
there
TK mRNA was not properly processed and become 785
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AND BIOPHYSICAL
the mature TK mRNA in the differentiated processing
RESEARCH COMMUNICATIONS
cells. The alteration
of this
event together with the result of the normal decay of TK mRNA may
explain the reduction It is interesting
of mature TK mRNA level in these differentiated
cells.
to note that Gudas et al. [17) have found that several nuclear
TK mRNA precursors with molecular
sizes at 8.8, 7.1, 6.4, 3.4 , 2.5 and 1.4 Kb
were present in the S phase of Balb/c 3T3 cells but were absent in quiescent cells.
They have suggested
that the lack of TK hnRNA processing
somehow signals the TK hnRNA degradation for the greatly diminished
in the nucleus, which may account
levels of mature TK RNA in quiescent
cells. Our results support their notion to some extent. findings suggested
the expression
HL-60 cells may be regulated
in G, cells
Balb/c 3T3
Taken together, these
of TK mRNA in monocytic-differentiated
by a post-transcriptional
control involved in
hnRNA splicing and turn over mechanisms.
ACKNOWLEDGMENTS This investigation was supported in part by grant CMRP262 from Chang Gung Medical College and a grant from NSC79-0412-Bl82-15 . I thank MS Duen-Yi Huang and Chiow-Jiau Lin for their able technical help and Dr. J.-K. Chen for his valuable advice. I am also grateful to Dr. Prescott L. Deninger and Dr. L. N. Wei for providing cDNA probes.
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