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Loss of collagenase gene expression in immortalized clones of SV40 T antigen-transformed human diploid fibroblasts
Vol. 189, No. 1, 1992 November 30, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 148-153
LOSS OF COLLAGENASE GENE EXPRESSION IN IMMORTALIZED CLONES OF SV40 T ANTIGEN-TRANSFORMED HUMAN DIPLOID FIBROBLASTS
Shin-ichiro Imai’ and Toshiya Takano
Department of Microbiology, Keio University School of Medicine, Tokyo 160, Japan Received
September
29,
1992
SUMMARY: We isolated a cDNA clone whose expression was lost during immortalization. The subtractive hybridization was performed between a genetically matched pair of mortal and immortal lines of SV40 T antigen-transformed MRC-5. The clone was found to code human interstitial collagenase. The expression of collagenase gene was almost completely shut off in seven out of eight independent immortalized clones. In addition, the levels of collagenase expression were dramatically increased toward crisis in the T antigen-transformed but mortal cells. These findings suggest the possibility that the regulatory mechanism of collagenase exB 1992Academic pression is related to both processes of in vitro aging and immortalization. Press,1°C.
The in vitro lifespan of human diploid fibroblasts (HDF) is so strictly controlled that HDF can hardly overcome the process of growth arrest termed cellular senescence (1, 2, 3). Oncogene products of DNA tumor viruses such as SV40 large T antigen and adenovirus ElA protein can induce immortalization of HDF at extremely low frequencies (4, 5). However, most of HDF transformed with those oncogene products fall into crisis after certain extended population doublings. Wright et al. (6) proposed the two-stage model: the first stage of cellular senescence which can be overcome by the function of large T antigen, and the second stage of crisis whose dysfunction results in immortalization.
Cell fusion experiments between different
immortalized cell lines suggest that a limited number of gene functions are lost in the immortalized cells (7). However, the molecules essential for immortalization, even the molecules that can be the hallmarks common to immortalized cells, are yet unknown.
To study the molecular basis of immortalization, genetically matched pairs of mortal and immortal cell clones should be used. We established T antigen-transformed HDF clones and their immortalized derivatives, and tried to isolate cDNA clones whose expression was completely lost in the immortalized cell clones, as the immortality
is reported to be a recessive
‘To whom correspondence should be addressed. Abbreviations: HDF, human diploid fibroblasts; PDL, population doubling level. 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
148
Vol.
189,
No.
1, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
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phenotype (8). The present study reports for the first time that the expression of human interstitial collagenase gene was almost completely lost after immortalization
in the SV40 large T-
transformed HDF. MATERIALS AND METHODS Cell Culture. MRC-5 (9) and its derivatives were cultured in a 1:l mixture of basal medium Eagle with Hank’s salts and that with Earle’s salts (Flow Laboratories, U.K.) supplemented with 10% fetal bovine serum (Hyclone Laboratories, UT). The cumulative numbers of population doubling level (PDL) were calculated from the cell numbers before and after splitting at each passage. Preparation of Subtracted cDNA Probe. Total RNA was isolated from the subconfluent cultures of a pre-immortalized clone HuS-L23 at PDL 69 to 70 as described (10). HuS-L23 entered into crisis at PDL 75. Poly(A)+ RNA was purified from the total RNA with Uligotex dT-30 (Takara Shuzo, Japan). [32P]dCTP-labeled cDNA was synthesized from the poly(A)+ RNA with oligo(dT) -18 primer by reverse transcriptase ( Superscript , Bethesda Research Laboratories, MD). ?wo successive cycles of subtractive hybridization were performed on the “2P-labeled H&-L23 cDNA with 35 to 40 pg poly(A)+ RNA of an immortalized clone IML23-1 at PDL 94 to 100 as described (11). The first and second subtractions achieved Ret values of approximately 5,300 and 4,700 mole nucleotide set per liter, respectively. The residual single-strand cDNA molecules were separated by hydroxyapatite (Bio-Rad) chromatography equilibrated in 0.10 M sodium phosphate buffer @H 7.0)/0.1% SDS at 60°C in a water-jacketed column. The recovery of the single-stranded DNA was about 2% of the loaded DNA after the two cycles of subtraction. The exogenously added poly(A)+ RNA of neo gene transcribed from pSP64Aneo (12) was concentrated about 4- to 7.5- and 2-fold by the first and the second subtractive hybridization, respectively. Library Screening. The cDNA library of a pre-immortalized clone was prepared from the poly(A)+ RNA of HuS-L23 at PDL 69 to 70 by the ZAP-cDNA synthesis kit (Stratagene, CA). The probe for screening clones was synthesized from the subtracted single-strand HuS-L23 cDNA by the random primer method (11). Fifty thousand independent plaques were examined by the hybridization with the subtracted HuS-L23 cDNA and with the IML23-1 cDNA probes. Plaque hybridization was carried out using nylon filter (Amersham) according to the protocol recommended by the manufacturer. Three hundred thirty-seven cDNA clones were initially picked and purified. The isolated plaques were individually gridded and screened by hybridization on duplicate sheets of nylon filter with 32P-labeled HuS-L23 and IML23-1 cDNA probes. After the second screening, 65 cDNA clones were isolated. Cross hybridization tests were done on the inserts to avoid redundant clones. Sequencing. Sequence analysis was done according to the cycle-sequencing protocol recommended by the manufacturer with an automated DNA sequencing system 373A (Applied Biosystems, CA). Sequences were compared with the data from the EMBL and the GenBank databases for homologies to known DNA sequences. Northern and Southern blot analyses. Five micrograms of total cellular RNA or 10 pg of the genomic DNA were analyzed by Northern or Southern blot analyses under stringent conditions of hybridization as described (11). The probes were synthesized from the inserts of cDNA clones by the random primer method (13). RESULTS Isolation
of cDNA
whose expression
was lost during
immortalization
MRC-5, a cell strain of human embryonal diploid fibroblasts, was transfected at PDL 27 with a plasmid carrying origin-defective SV40 early gene (14). From two different mortal clones of T antigen-transformed MRC-5 designated as HuS-L23 and HuS-L12, we obtained eight independent immortalized lines. Three were derived from HuS-L23 and designated as IML23-1 to -3. Other five lines were obtained from HuS-L12 and designated as IML12-1 to -5.
149
Vol.
BIOCHEMICAL
189, No. 1, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Subtractive hybridization of HuS-L23 cDNA was conducted with excess poly(A)+ RNA of IML23-1
and this subtracted cDNA preparation was used as the probe for screening the
cDNA library of HuS-L23.
Three independent cDNA clones, designated as clone 103,909 and
2407, were isolated in 5 x lo4 HuS-L23 cDNA clones screened. One of the isolates, clone 2407, was found to carry the insert whose expression was almost completely lost in IML23-1 cells (Fig. 1). Clone 2407 was identified as the cDNA of human interstitial collagenase by sequence analysis and computer-aided
comparison with DNA database (data not shown).
Although 20 base pairs at the 5’ end of collagenase cDNA was deleted in clone 2407, the restriction map of the clone was coincident with that expected from the reported full-length collagenase cDNA (15). Other two cDNA clones isolated, clone 103 and 909, were found to code 27kDa heat shock protein and follistatin, respectively.
Because these clones did not show such
wide differences of expression in HuS-L23 and IML23-1
cells as those in the case of clone
2407 (Fig. l), we concentrated on the analysis of clone 2407. Collagenase expression in pre-immortalized
and immortalized cells
Collagenase gene was highly expressed in both of the pre-immortalized
clones HuS-
L12 and -L23 at the late PDLs (Fig. 2). However, the expression levels were greatly reduced in seven out of eight immortalized derivatives. The expression was totally undetectable in IML23-3,
IML12-4
and -5 (Fig. 2). Only by the longer exposure of autoradiography,
bands of collagenase mRNA were detected in IML23-1,
IML12-1,
faint
-2 and -3 (Fig. 2, middle
panels). The levels of expression were 21- to 63-fold less in these cells than those in the paren-
CLONE 103
CLONE 909
12
CLONE 2407
12
12
42.7kb
.Qkb
FIG. 1. Expression of the genes corresponding to clone 103,909 and 2407 in the pre-immortal-S-L23 (lane 1) and the immortalized IML23-1 (lane 2) cells. Five micrograms per lane of total cellular RNA was analyzed by Northern blot hybridization with the probes synthesized from the inserts of the clones. The acridine orange stains of the RNA samples used for the blots are shown in the bottom panels. Positions of 28s and 18s rRNA are indicated by short lines.
150
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BIOCHEMICAL
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
-
FIG. 2. Expression of collagenase gene in pre-immortalized and immortalized clones. Total cellular RNA of (A) L23 series and (B) L12 series was analyzed and the results are shown similarly as Fig. 1. The middle panels show longer exposures in autoradiography of the same blots as the top panels.
tal pre-immortalized
cells. Only in IML23-2,
collagenase expression was predominantly high
and the level was about twice of that in the parental HuS-L23. By Southern blot analysis, any significant differences were not detected in the collagenase gene structure of the pre-immortalized and immortalized cell clones, although extra weak bands of various sizes not found in MRC-5 were observed in these cell clones (Fig. 3). The 413-base pair upstream flanking sequences of collagenase gene did not carry any mutations in the immortalized clones (data not shown). Collagenase expression during in vitro aging In the pre-immortalized
HuS-L12 clone, the expression level was substantially low at
PDL 69. However, much more abundant collagenase mRNA was detected from PDL 76 and the expression level rose toward the crisis which occurred at PDL 92 (Fig. 4). At PDL 76, the amount of collagenase mRNA was twenty-fold 82 as much as twice of those at PDL 76.
of those at PDL 73, and still increased at PDL
DISCUSSION Our results clearly demonstrated that the level of collagenase mRNA dramatically shifted up and down during in vitro aging and immortalization. The T antigen-transformed but mortal cells showed a dramatic increase of the collagenase expression toward crisis. Once the cells immortalized, the expression was almost completely switched off. These results suggested that the regulation of collagenase gene was correlated with both processes of in vitro aging and 1.51
Vol.
189, No. 1, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
23 9.4 6.6 4.4
1.4 1.1 0.67 0.60
-
I:%
-',$::, -' " "f‘r'i,,.l;, .(( .(
1.4
i, ""1
0.: 0.60
FIG. 3. Southern blot analyses of collagenase gene in pm-immortalized and immortalized clones. Ten micrograms per lane of EcoRI-digested genomic DNA was analyzed on (A) L23 series and (B) L12 series by Southern blot hybridization with the 1.5-kilobase EcoRI-XhoI probe of the clone 2407. The positions of molecular markers were indicated at the left sides of the panels.
PDL
69
73
76
82
90
92
FIG. 4 Expression of collagenase gene during the process of in vitro aging in T antigen-transformed mortal clone HuS-L12. Total cellular RNA was analyzed and the results are shown as well as those in Fig. 1. Numbers on top indicate the PDLs of cells examined. HuS-L12 entered into crisis at PDL 92 in this experiment.
152
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BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
immortalization. Exceptionally, the immortalized clone IML23-2
showed the prominent col-
lagenase expression. Pereira-Smith and Smith (7) assigned immortalized cell lines of various origins to at least four complementation
groups. Their findings imply that multiple different
genes are involved in the process-of immortalization. talization of IML23-2
The genetic lesion that caused the immor-
may have been different from those that brought about the immortal
phenotypes to the other immortalized clones examined in this study. The loss of collagenase expression in the immortalized cells was unlikely due to the rearrangements or mutations occurred in the collagenase gene, because the coding and the regulatory regions of collagenase gene were not changed in these cells. Collagenase mRNA is reported to be stable (16). Thus, a certain trans-acting machinery seemed to be involved in the drastic changes of collagenase expression in the pre-immortalized
and immortalized cells. SV40 T
antigen might participate in the machinery. However, it is more likely that an unknown factor other than T antigen is responsible for the loss of collagenase expression, because of the following reasons. First, the amounts of T antigen and its binding to retinoblastoma gene product and ~53 were at similar levels in the immortalized cells as those in the parental pre-immortalized cells (our unpublished data). Second, the expression of exogenously introduced T antigen gene did not increase the level of collagenase mRNA in IML23-1
cells (our unpublished finding).
Third, the function of T antigen is reported to be necessary but insufficient for immortalization (6, 17). The hypothetical factor responsible for the loss of collagenase expression may be implicated in the immortalization process of T antigen-transformed HDF. ACKNOWLEDGMENTS We thank Dr. Michael Karin and his colleagues for many critical discussions, Akihiro Umezawa and Tadahiro Fujino for their help in subtractive hybridization. We also thank Minoru S. H. Ko for his gift of pSP64Aneo. REFERENCES ;: 3. 4. 5. 6. 7. t. Id. 11.
::: 14. 15. 16. 17.
Hayflick, L. and Moorhead, P.S. (1961) Exp. Cell Res. 25,585-621. Stanulis-Praeger, B.M. (1987) Mech. Ageing Dev. 38, l-48. Goldstein, S. (1990) Science 249, 1129-1133. Shay, J.W. and Wright, W.E. (1989) Exp. Cell Res. 184,109-118. Shay, J.W., Wright, W.E., and Werbin, H. (1991) Biochim. Biophys. Acta 1072,1-7. Wright, W.E., Pereira-Smith, O.M., and Shay, J.W. (1989) Mol. Cell. Biol. 9, 3088-3092. Pereira-Smith, O.M. and Smith, J.R. (1988) Proc. Natl. Acad. Sci. USA 85, 6042-6046. Pereira-Smith, O.M. and Smith, J.R. (1983) Science 221,964-966. Jacobs, J.P., Jones, C.M., and Baille, J.P. (1970) Nature 227, 168-170. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry 18,5294-5299. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (second edition). (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Ko, M.S.H. (1990) Nucleic Acids Res. 18,5705-5711. Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. Small, M.B., Gluzman, Y., and Ozer, H.L. (1982) Nature 296,671-672. Whitham, S.E., Murphy, G., Angel, P., Rahmsdorf, H.-J., Smith, B.J., Lyons, A., Harris, T.J.R., Reynolds, J.J., Herrlich, P., and Docherty, A.J.P. (1986) Biochem. J. 240, 913-916. Angel, P., Baumann, I., Stein, B., Delius, H., Rahmsdorf, H.J., and Herrlich, P. (1987) Mol. Cell. Biol. 7,2256-2266. Neufeld, D.S., Riply, S., Henderson, A., and Ozer, H.L. (1987) Mol. Cell. Biol. 7, 2794-2802. 153
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 148-153
LOSS OF COLLAGENASE GENE EXPRESSION IN IMMORTALIZED CLONES OF SV40 T ANTIGEN-TRANSFORMED HUMAN DIPLOID FIBROBLASTS
Shin-ichiro Imai’ and Toshiya Takano
Department of Microbiology, Keio University School of Medicine, Tokyo 160, Japan Received
September
29,
1992
SUMMARY: We isolated a cDNA clone whose expression was lost during immortalization. The subtractive hybridization was performed between a genetically matched pair of mortal and immortal lines of SV40 T antigen-transformed MRC-5. The clone was found to code human interstitial collagenase. The expression of collagenase gene was almost completely shut off in seven out of eight independent immortalized clones. In addition, the levels of collagenase expression were dramatically increased toward crisis in the T antigen-transformed but mortal cells. These findings suggest the possibility that the regulatory mechanism of collagenase exB 1992Academic pression is related to both processes of in vitro aging and immortalization. Press,1°C.
The in vitro lifespan of human diploid fibroblasts (HDF) is so strictly controlled that HDF can hardly overcome the process of growth arrest termed cellular senescence (1, 2, 3). Oncogene products of DNA tumor viruses such as SV40 large T antigen and adenovirus ElA protein can induce immortalization of HDF at extremely low frequencies (4, 5). However, most of HDF transformed with those oncogene products fall into crisis after certain extended population doublings. Wright et al. (6) proposed the two-stage model: the first stage of cellular senescence which can be overcome by the function of large T antigen, and the second stage of crisis whose dysfunction results in immortalization.
Cell fusion experiments between different
immortalized cell lines suggest that a limited number of gene functions are lost in the immortalized cells (7). However, the molecules essential for immortalization, even the molecules that can be the hallmarks common to immortalized cells, are yet unknown.
To study the molecular basis of immortalization, genetically matched pairs of mortal and immortal cell clones should be used. We established T antigen-transformed HDF clones and their immortalized derivatives, and tried to isolate cDNA clones whose expression was completely lost in the immortalized cell clones, as the immortality
is reported to be a recessive
‘To whom correspondence should be addressed. Abbreviations: HDF, human diploid fibroblasts; PDL, population doubling level. 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
148
Vol.
189,
No.
1, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
phenotype (8). The present study reports for the first time that the expression of human interstitial collagenase gene was almost completely lost after immortalization
in the SV40 large T-
transformed HDF. MATERIALS AND METHODS Cell Culture. MRC-5 (9) and its derivatives were cultured in a 1:l mixture of basal medium Eagle with Hank’s salts and that with Earle’s salts (Flow Laboratories, U.K.) supplemented with 10% fetal bovine serum (Hyclone Laboratories, UT). The cumulative numbers of population doubling level (PDL) were calculated from the cell numbers before and after splitting at each passage. Preparation of Subtracted cDNA Probe. Total RNA was isolated from the subconfluent cultures of a pre-immortalized clone HuS-L23 at PDL 69 to 70 as described (10). HuS-L23 entered into crisis at PDL 75. Poly(A)+ RNA was purified from the total RNA with Uligotex dT-30 (Takara Shuzo, Japan). [32P]dCTP-labeled cDNA was synthesized from the poly(A)+ RNA with oligo(dT) -18 primer by reverse transcriptase ( Superscript , Bethesda Research Laboratories, MD). ?wo successive cycles of subtractive hybridization were performed on the “2P-labeled H&-L23 cDNA with 35 to 40 pg poly(A)+ RNA of an immortalized clone IML23-1 at PDL 94 to 100 as described (11). The first and second subtractions achieved Ret values of approximately 5,300 and 4,700 mole nucleotide set per liter, respectively. The residual single-strand cDNA molecules were separated by hydroxyapatite (Bio-Rad) chromatography equilibrated in 0.10 M sodium phosphate buffer @H 7.0)/0.1% SDS at 60°C in a water-jacketed column. The recovery of the single-stranded DNA was about 2% of the loaded DNA after the two cycles of subtraction. The exogenously added poly(A)+ RNA of neo gene transcribed from pSP64Aneo (12) was concentrated about 4- to 7.5- and 2-fold by the first and the second subtractive hybridization, respectively. Library Screening. The cDNA library of a pre-immortalized clone was prepared from the poly(A)+ RNA of HuS-L23 at PDL 69 to 70 by the ZAP-cDNA synthesis kit (Stratagene, CA). The probe for screening clones was synthesized from the subtracted single-strand HuS-L23 cDNA by the random primer method (11). Fifty thousand independent plaques were examined by the hybridization with the subtracted HuS-L23 cDNA and with the IML23-1 cDNA probes. Plaque hybridization was carried out using nylon filter (Amersham) according to the protocol recommended by the manufacturer. Three hundred thirty-seven cDNA clones were initially picked and purified. The isolated plaques were individually gridded and screened by hybridization on duplicate sheets of nylon filter with 32P-labeled HuS-L23 and IML23-1 cDNA probes. After the second screening, 65 cDNA clones were isolated. Cross hybridization tests were done on the inserts to avoid redundant clones. Sequencing. Sequence analysis was done according to the cycle-sequencing protocol recommended by the manufacturer with an automated DNA sequencing system 373A (Applied Biosystems, CA). Sequences were compared with the data from the EMBL and the GenBank databases for homologies to known DNA sequences. Northern and Southern blot analyses. Five micrograms of total cellular RNA or 10 pg of the genomic DNA were analyzed by Northern or Southern blot analyses under stringent conditions of hybridization as described (11). The probes were synthesized from the inserts of cDNA clones by the random primer method (13). RESULTS Isolation
of cDNA
whose expression
was lost during
immortalization
MRC-5, a cell strain of human embryonal diploid fibroblasts, was transfected at PDL 27 with a plasmid carrying origin-defective SV40 early gene (14). From two different mortal clones of T antigen-transformed MRC-5 designated as HuS-L23 and HuS-L12, we obtained eight independent immortalized lines. Three were derived from HuS-L23 and designated as IML23-1 to -3. Other five lines were obtained from HuS-L12 and designated as IML12-1 to -5.
149
Vol.
BIOCHEMICAL
189, No. 1, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Subtractive hybridization of HuS-L23 cDNA was conducted with excess poly(A)+ RNA of IML23-1
and this subtracted cDNA preparation was used as the probe for screening the
cDNA library of HuS-L23.
Three independent cDNA clones, designated as clone 103,909 and
2407, were isolated in 5 x lo4 HuS-L23 cDNA clones screened. One of the isolates, clone 2407, was found to carry the insert whose expression was almost completely lost in IML23-1 cells (Fig. 1). Clone 2407 was identified as the cDNA of human interstitial collagenase by sequence analysis and computer-aided
comparison with DNA database (data not shown).
Although 20 base pairs at the 5’ end of collagenase cDNA was deleted in clone 2407, the restriction map of the clone was coincident with that expected from the reported full-length collagenase cDNA (15). Other two cDNA clones isolated, clone 103 and 909, were found to code 27kDa heat shock protein and follistatin, respectively.
Because these clones did not show such
wide differences of expression in HuS-L23 and IML23-1
cells as those in the case of clone
2407 (Fig. l), we concentrated on the analysis of clone 2407. Collagenase expression in pre-immortalized
and immortalized cells
Collagenase gene was highly expressed in both of the pre-immortalized
clones HuS-
L12 and -L23 at the late PDLs (Fig. 2). However, the expression levels were greatly reduced in seven out of eight immortalized derivatives. The expression was totally undetectable in IML23-3,
IML12-4
and -5 (Fig. 2). Only by the longer exposure of autoradiography,
bands of collagenase mRNA were detected in IML23-1,
IML12-1,
faint
-2 and -3 (Fig. 2, middle
panels). The levels of expression were 21- to 63-fold less in these cells than those in the paren-
CLONE 103
CLONE 909
12
CLONE 2407
12
12
42.7kb
.Qkb
FIG. 1. Expression of the genes corresponding to clone 103,909 and 2407 in the pre-immortal-S-L23 (lane 1) and the immortalized IML23-1 (lane 2) cells. Five micrograms per lane of total cellular RNA was analyzed by Northern blot hybridization with the probes synthesized from the inserts of the clones. The acridine orange stains of the RNA samples used for the blots are shown in the bottom panels. Positions of 28s and 18s rRNA are indicated by short lines.
150
Vol.
BIOCHEMICAL
189, No. 1, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
-
FIG. 2. Expression of collagenase gene in pre-immortalized and immortalized clones. Total cellular RNA of (A) L23 series and (B) L12 series was analyzed and the results are shown similarly as Fig. 1. The middle panels show longer exposures in autoradiography of the same blots as the top panels.
tal pre-immortalized
cells. Only in IML23-2,
collagenase expression was predominantly high
and the level was about twice of that in the parental HuS-L23. By Southern blot analysis, any significant differences were not detected in the collagenase gene structure of the pre-immortalized and immortalized cell clones, although extra weak bands of various sizes not found in MRC-5 were observed in these cell clones (Fig. 3). The 413-base pair upstream flanking sequences of collagenase gene did not carry any mutations in the immortalized clones (data not shown). Collagenase expression during in vitro aging In the pre-immortalized
HuS-L12 clone, the expression level was substantially low at
PDL 69. However, much more abundant collagenase mRNA was detected from PDL 76 and the expression level rose toward the crisis which occurred at PDL 92 (Fig. 4). At PDL 76, the amount of collagenase mRNA was twenty-fold 82 as much as twice of those at PDL 76.
of those at PDL 73, and still increased at PDL
DISCUSSION Our results clearly demonstrated that the level of collagenase mRNA dramatically shifted up and down during in vitro aging and immortalization. The T antigen-transformed but mortal cells showed a dramatic increase of the collagenase expression toward crisis. Once the cells immortalized, the expression was almost completely switched off. These results suggested that the regulation of collagenase gene was correlated with both processes of in vitro aging and 1.51
Vol.
189, No. 1, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
23 9.4 6.6 4.4
1.4 1.1 0.67 0.60
-
I:%
-',$::, -' " "f‘r'i,,.l;, .(( .(
1.4
i, ""1
0.: 0.60
FIG. 3. Southern blot analyses of collagenase gene in pm-immortalized and immortalized clones. Ten micrograms per lane of EcoRI-digested genomic DNA was analyzed on (A) L23 series and (B) L12 series by Southern blot hybridization with the 1.5-kilobase EcoRI-XhoI probe of the clone 2407. The positions of molecular markers were indicated at the left sides of the panels.
PDL
69
73
76
82
90
92
FIG. 4 Expression of collagenase gene during the process of in vitro aging in T antigen-transformed mortal clone HuS-L12. Total cellular RNA was analyzed and the results are shown as well as those in Fig. 1. Numbers on top indicate the PDLs of cells examined. HuS-L12 entered into crisis at PDL 92 in this experiment.
152
Vol.
189, No. 1, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
immortalization. Exceptionally, the immortalized clone IML23-2
showed the prominent col-
lagenase expression. Pereira-Smith and Smith (7) assigned immortalized cell lines of various origins to at least four complementation
groups. Their findings imply that multiple different
genes are involved in the process-of immortalization. talization of IML23-2
The genetic lesion that caused the immor-
may have been different from those that brought about the immortal
phenotypes to the other immortalized clones examined in this study. The loss of collagenase expression in the immortalized cells was unlikely due to the rearrangements or mutations occurred in the collagenase gene, because the coding and the regulatory regions of collagenase gene were not changed in these cells. Collagenase mRNA is reported to be stable (16). Thus, a certain trans-acting machinery seemed to be involved in the drastic changes of collagenase expression in the pre-immortalized
and immortalized cells. SV40 T
antigen might participate in the machinery. However, it is more likely that an unknown factor other than T antigen is responsible for the loss of collagenase expression, because of the following reasons. First, the amounts of T antigen and its binding to retinoblastoma gene product and ~53 were at similar levels in the immortalized cells as those in the parental pre-immortalized cells (our unpublished data). Second, the expression of exogenously introduced T antigen gene did not increase the level of collagenase mRNA in IML23-1
cells (our unpublished finding).
Third, the function of T antigen is reported to be necessary but insufficient for immortalization (6, 17). The hypothetical factor responsible for the loss of collagenase expression may be implicated in the immortalization process of T antigen-transformed HDF. ACKNOWLEDGMENTS We thank Dr. Michael Karin and his colleagues for many critical discussions, Akihiro Umezawa and Tadahiro Fujino for their help in subtractive hybridization. We also thank Minoru S. H. Ko for his gift of pSP64Aneo. REFERENCES ;: 3. 4. 5. 6. 7. t. Id. 11.
::: 14. 15. 16. 17.
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