©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Autocrine Transforming Growth Factor Modulates the Expression of Integrin in Human Colon Carcinoma FET Cells (*)

DanHui Wang (1)(§)(¶), Guo-hao Zhou(¶) (2)(**), Thomas M. Birkenmeier (3), Jiangen Gong (1)(§), LuZhe Sun (1), Michael G. Brattain (1)(§§)

From the (1) Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43699-0008, the (2) Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, and the (3) Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor (TGF-) has been extensively studied as an exogenous agent that stimulates the expression of extracellular matrix proteins and their cell-surface integrin receptors in a variety of cell types. However, the recent demonstration of autocrine TGF- growth effects in a number of cell types suggests that the steady-state expression of extracellular matrix and integrin proteins and their biological activity may also be under autocrine TGF- control. Previously, we reported that repression of autocrine TGF- activity by constitutive expression of a full-length TGF- antisense cDNA led to abrogation of autocrine negative TGF- and, as a result, increased tumorigenicity and anchorage-independent growth of a poorly tumorigenic, well-differentiated colon carcinoma cell line designated FET (Wu, S., Theodorescu, D., Kerbel, R. S., Willson, J. K. V., Mulder, K. M., Humphrey, L. E., and Brattain, M. G.(1992) J. Cell Biol. 116, 187-196). Consequently, we have used this model system to study the effects of repression of autocrine TGF- activity on the expression of integrin and integrin -mediated cell adhesion to fibronectin. The expression of the integrin subunit was reduced in TGF- antisense transfected FET cells at both mRNA and protein levels as determined by RNase protection assays and immunoprecipitation, respectively. Autocrine TGF- had no effect on the transcription of integrin and subunits, indicating that autocrine TGF- may regulate integrin expression at the post-transcriptional level. The diminished expression of integrin on the cell surface led to the reduced adhesion of TGF- antisense transfected cells to fibronectin. This phenomenon could be reversed by treatment with exogenous TGF-.


INTRODUCTION

Cell adhesion to the extracellular matrix (ECM),() mediated in part by the integrin family of cell-surface glycoproteins, plays an important role in the control of cell migration, proliferation, differentiation, and invasion (1, 2). Integrins are formed by the noncovalent association of an subunit with a subunit. The subunit dimerized by at least six subunits, termed -, constitutes the largest component of the integrin family. Among them, , , and are the receptors for collagen, laminin, and other ECM proteins, whereas is the receptor specific for FN.

Integrins have an important biological role as mediators of cell matrix-cell interaction. A number of studies have demonstrated that integrins are associated with transformation and differentiation of certain cell types. For example, overexpression of integrin reverses tumorigenicity and anchorage-independent growth in transformed Chinese hamster ovary cells (3). Similar results were obtained in a stable variant of the K562 erythroleukemia cell line that overexpresses integrin (4) . In contrast, loss of integrin expression is associated with a transformed phenotype of fibroblasts (5) , and increased tumorigenicity was shown in integrin -deficient Chinese hamster ovary cell variants (6) . The integrins also have a signal transduction function that affects the expression of many essential genes relevant to matrix remodeling during differentiation. For example, monoclonal antibody to integrin induces the expression of two extracellular matrix-degrading metalloproteinases, collagenase and stromelysin (7) . Recently, we showed that antibody to leads to increased DNA synthesis in quiescent HT1080 cells (8) .

The role of integrins in a wide array of biological processes underlines the significance of determining the mechanisms by which particular cell types control the steady-state expression of these receptors as well as the mechanisms by which the extracellular environment can alter their expression. Extensive studies have shown that the expression of integrins is under the control of several growth factors and cytokines. For example, platelet-derived growth factor BB was found to induce integrin and subunit expression in rabbit vascular smooth muscle cells (9) , and interleukin-1 was shown to increase integrin and subunit mRNA levels, but to decrease integrin subunit mRNA levels in MG-63 human osteosarcoma cells (10). Among the growth factors, TGF- has been the most extensively studied exogenous modulator of the expression of ECM proteins and their integrin receptors (11) . TGF- has been shown to stimulate the expression of FN and collagen and their incorporation into the ECM (12). A number of integrin subunits in the subfamily have been shown to be stimulated by TGF- in human fibroblasts (13, 14) . Treatment of MG-63 human osteosarcoma cells with TGF- markedly decreases integrin subunit mRNA and protein levels with concomitant increases in , , and subunit expression (15) . Interestingly, the Engelbreth-Holm-Swarm matrix was found to down-regulate TGF- synthesis at the transcriptional level in mouse epithelial cells (16) .

As indicated above, most studies of TGF- function and mechanism have centered around treatment of cells with one of the TGF- isoforms. However, it is generally believed that TGF- exerts its role in an autocrine as well as a paracrine fashion (17) . The autocrine negative growth activity of TGF- was demonstrated by the ability of neutralizing TGF- and TGF- antibodies to increase proliferation, DNA synthesis, and anchorage-independent growth (18, 19, 20, 21) . We have used stable transfection of a TGF- antisense cDNA to demonstrate autocrine TGF- activity in a colon carcinoma cell line designated FET (21) . Characterization of transfected FET cells with constitutively repressed TGF- expression showed high cloning efficiency in anchorage-independent assays and enhanced tumorigenicity in athymic nude mice. These studies suggested that loss of autocrine TGF- activity may be an important step in progression of malignancy. Given the effects of exogenous TGF- treatment on the expression of integrins in a wide variety of model systems, we hypothesized that autocrine TGF- may control biological function by regulating steady-state integrin receptor expression and consequently cellular interactions with the ECM. We have tested this hypothesis using the FET model system, described above, in which autocrine TGF- activity was constitutively repressed (21) .


MATERIALS AND METHODS

Cells and Culture

The FET human colon carcinoma cell line was originally established in vitro from a primary human colon tumor (22) . FET TGF- antisense transfected cells (FET B) and FET control cells transfected with the neomycin-resistant gene were established and characterized as described previously (21) . Cells were maintained in chemically defined McCoy's 5A serum-free medium (Life Technologies, Inc.) supplemented with 20 µg/ml insulin (Sigma), 4 µg/ml transferrin (Sigma), and 10 ng/ml epidermal growth factor (Sigma) at 37 °C in a humidified atmosphere of 5% CO as described previously (23) . Subcultures were obtained by treatment with 0.125% trypsin in Joklik's tissue culture medium (Life Technologies, Inc.) containing 0.1% EDTA.

RNase Protection Assays

Total RNA was isolated from cultured cells by the guanidine isothiocyanate method (24) . RNase protection assays were performed as described by Wu et al. (25). The subunit template was constructed by subcloning a 405-base pair BamHI-AccI fragment of the human subunit cDNA into plasmid pBSK (Stratagene cloning system). A 214-base pair SpeI-PstI fragment of integrin cDNA was also inserted into the pBSK vector. Both and subunit antisense probes were prepared using T RNA polymerase. The construction of the TGF- antisense probe has been described previously by Wu et al.(21) . Determinations of the integrin subunit and TGF- mRNA levels in total RNA samples (40 µg) were performed with 1 10 cpm of each of the labeled probes. The hybridization mixture was digested with RNase A and RNase T, followed by treatment with proteinase K. The protected fragment of the probe was analyzed by urea-polyacrylamide gel electrophoresis and visualized by autoradiography.

Immunoprecipitation

To quantitate the cell-surface subunit protein level, 10 cells were resuspended in 1 ml of phosphate-buffered saline (pH 7.4), and cell-surface proteins were labeled with 0.1 mg/ml sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce). The labeled cells were washed with phosphate-buffered saline and solubilized with 1 ml of extraction buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM CaCl, 1 mM MgCl, 1% Nonidet P-40). Cell extract (300 µl) was precleared with 30 µl of protein G-agarose suspension (Pierce) for 8 h at 4 °C. Biotinylated integrin was immunoprecipitated with 2 µl of monoclonal anti-human integrin antibody (Life Technologies, Inc.) and 50 µl of protein G-agarose after overnight incubation at 4 °C. The precipitates were washed six times with buffer containing 50 mM Tris (pH 7.5), 0.5 M NaCl, 1 mM CaCl, 1 mM MgCl, and 0.1% Tween 20. The biotinylated and subunits were dissociated from the immune complex by heating the samples in SDS nonreducing sample buffer at 95 °C for 10 min and were analyzed by SDS-polyacrylamide gel electrophoresis using a Bio-Rad Mini-PROTEAN II gel apparatus. The proteins were electrotransferred onto an Immun-Lite blotting membrane (Bio-Rad), which was then blocked with 5% nonfat dry milk and incubated for 1 h in the presence of streptavidin-alkaline phosphatase (Life Technologies, Inc.). A chemiluminescent substrate, 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (Bio-Rad), for alkaline phosphatase was then added to the blot. The and subunits were visualized after autoradiography.

Transient Transfection and CAT Assay

The subunit promoter-CAT constructs used in this study have been reported previously (26) . Promoter activity was measured using the P-924 CAT and P-1300 CAT constructs containing 924 and 1300 base pairs, respectively, 5` of the transcription start site. FET Neo control and TGF- antisense transfected cells were transfected with 30 µg of promoter-CAT plasmids together with 10 µg of -galactosidase plasmid by electroporation with a Bio-Rad Gene Pulser at 250 V and 960 microfarads. The electroporated cells were plated onto 10-cm culture dishes in the serum-free medium and harvested after 48 h. The expression of the CAT reporter gene was assayed to determine the promoter activity in these cells. The cell pellets were resuspended in 0.25 M Tris (pH 7.8) and lysed by three cycles of freeze-thaw. -Galactosidase activity in the cell extracts was quantified to normalize the amounts of extracts for CAT assay. Aliquots representing equal amounts of -galactosidase activity were incubated with [C]chloramphenicol (0.25 µCi) and acetyl coenzyme A (114 µg) for 6 h at 37 °C, extracted with ethyl acetate, and separated by thin-layer chromatography as described by Sambrook et al.(27) . The levels of acetylated chloramphenicol were quantitated by the AMBIS image acquisition and analysis system. Isolation of nuclei and nuclear run-on assays were performed essentially as described by Greenberg and Ziff (28) with some modifications (29) .

Cell Adhesion and MTT Assays

Ninety-six-well Corning tissue culture plates were coated with FN at concentrations of 0, 0.1, 0.3, 0.6, 1.2, 2.4, 5.0, and 10.0 µg/ml for 2 h at 37 °C and then rinsed once with phosphate-buffered saline. Confluent cells were detached by treatment with trypsin, plated at 6 10 cells/well on FN-coated plates, and incubated for 90 min in a humidified incubator. Unattached cells were gently washed away by four rinses with phosphate-buffered saline, and attached cells were determined by the MTT assay as described previously (30, 31) .

The specificity of cell adhesion to FN was determined using the monoclonal anti-human integrin subunit antibody. The antibody was added to FN-coated plates at dilutions of 1:50-1:500 and incubated for 30 min at 37 °C. Determination of cell adhesion and the MTT assay were performed as described above.


RESULTS

Exogenous TGF-Increases Integrin Subunit mRNA Steady-state Level in FET Parental Cells

The FET cell line is a poorly tumorigenic, well-differentiated human colon carcinoma cell line that was previously established in vitro from a primary tumor and has been extensively characterized (22, 32) . FET cells expressed high levels of TGF- mRNA, but no detectable TGF- and TGF- mRNAs by Northern blot analysis (21) . To determine whether exogenous TGF- can stimulate integrin subunit expression in FET parental cells, the steady-state levels of mRNA from the cells treated with TGF- (5 ng/ml) at different time points were compared by RNase protection assay (Fig. 1). The steady-state mRNA level of integrin subunit was increased after 12 h of exposure to TGF-, while a maximal increase was observed after 24 h of TGF- treatment.


Figure 1: Effect of exogenous TGF- on integrin subunit mRNA levels in FET cells. Total RNA was extracted from FET cells treated with TGF- (5 ng/ml) for 0, 2, 6, 12, 24, and 48 h. Integrin subunit mRNA and actin mRNA were detected in 40 µg of total RNA using a RNase protection assay. Actin mRNA levels are shown to indicate equal loading of the samples. Molecular weight markers are shown in lane M.



Repression of Autocrine TGF- Activity Reduces Integrin Subunit mRNA Steady-state Level and Cell-surface Integrin Expression

Repressed autocrine TGF- activity was obtained by constitutive expression of a TGF- antisense cDNA in FET cells. A typical stable clone, designated FET B, that expresses high levels of TGF- antisense cDNA was selected for these studies (21) . The steady-state mRNA levels of integrin and subunits in FET B and FET Neo control cells were determined by RNase protection analysis (Fig. 2). FET B cells showed an 5-fold reduction of the TGF- mRNA level, as published previously (21) . The expression of integrin subunit mRNA was reduced 2.5-fold in FET B cells compared with FET Neo cells. The modulation of expression was relatively specific in that integrin subunit mRNA expression was not altered by repression of autocrine TGF-. In addition, there was no change in the mRNA levels of integrin and subunits (data not shown). Reduced mRNA expression suggested a corresponding reduction of receptor expression on the cell surface of FET B cells compared with FET Neo cells. This was confirmed by immunoprecipitation experiments examining the cell-surface expression of integrin (Fig. 3).


Figure 2: Integrin and mRNA levels in FET cells transfected with TGF- antisense expression plasmid (FET B) and control plasmid (FET Neo). Total RNA was isolated from both cell lines. Integrin and subunit mRNAs, TGF- mRNA, and actin mRNA were detected in 40 µg of total RNA by a RNase protection assay. The two photographs are from one gel. Because of the low expression of the integrin subunit, the upper photograph was exposed for a longer period of time than the lower one. Actin mRNA levels are shown to indicate equal loading of the samples.




Figure 3: Cell-surface integrin protein levels in FET cells transfected with TGF- antisense cDNA (FET B) and control plasmid (FET Neo). Cell-surface proteins from equal numbers of cells were labeled with biotin. Biotinylated integrin was immunoprecipitated with an anti-human subunit antibody and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. The negative control did not receive anti- antibody during immunoprecipitation.



Autocrine TGF- Modulates Integrin Expression at the Post-transcriptional Level

To examine whether the down-regulation of integrin expression by repression of autocrine TGF- was due to transcriptional regulation, we transiently transfected integrin subunit promoter-CAT constructs (26) into FET B and FET Neo cells. The levels of CAT enzyme, expressed under the control of the integrin promoter, were quantitated using the standard assay for [C]chloramphenicol conversion (Fig. 4). There was no difference in the CAT activity in FET B and FET Neo cells after transfection with the CAT constructs. A nuclear run-on assay was performed to confirm these results (data not shown). Again, no difference in the subunit transcription rate was observed in FET B and FET Neo cells. These results indicate that autocrine TGF- does not regulate integrin expression at transcriptional levels.


Figure 4: Integrin promoter (924- and 1300-base pair fragments) activity in FET cells transfected with TGF- antisense expression plasmid (FET B) and control plasmid (FET Neo). A, FET B and FET Neo cells were transfected with P-924 CAT and P-1300 CAT plasmids. Cells were harvested 48 h after transfection. -Galactosidase activity in cell extracts was normalized for CAT assay. B, the amounts of product and remaining substrate were quantitated by scanning the autoradiographic film. The graph was plotted by the ratio of product to substrate.



Reduction of Cell-surface Integrin Expression Leads to Reduced Cell Adhesion to FN That Can be Reversed by Exogenous TGF-Treatment

Next, it was determined whether the down-regulation of expression affected biological function as reflected by alterations of cell adhesion to FN (Fig. 5). Levels of enhancement ranged from 1.4 to 2.8-fold for FET Neo cells when coating was performed at FN concentrations ranging from 0.1 to 10 µg/ml, while FN coating had little effect on FET B cell adhesion as an enhancement of only 1.4-fold over bovine serum albumin coating was obtained at a FN concentration of 10 µg/ml (Fig. 5A). Thus, adhesion to FN-coated culture plates relative to bovine serum albumin-coated plates was enhanced 4-5-fold for FET Neo cells relative to FET B cells in the FN range between 1.2 and 10 µg/ml. Treatment of both FET Neo and FET B cells with 5 ng/ml TGF- increased the level of binding to FN-coated plates over bovine serum albumin controls (Fig. 5B). After TGF- treatment, the level of enhancement of adhesion to FN for both cell types was approximately equal. These results indicate that repression of autocrine TGF- leads to reduced adhesion to FN. Moreover, treatment with exogenous TGF- led to restoration of binding to FN. The reduced levels of adhesion in FET B cells were consistent with the lower steady-state levels of cell-surface integrin in these cells relative to control FET Neo cells. We hypothesized that restoration of adhesion after TGF- treatment of FET B cells was due to induction of integrin expression. Consequently, the concentration-dependent effects of TGF- treatment on cell-surface integrin expression were determined by immunoprecipitation in both types of cells. TGF- treatment increased both FET Neo and FET B expression of cell-surface integrin (data not shown).


Figure 5: Adhesion of FET control () and FET TGF- antisense transfected () cells as a function of FN concentration. Substrates were prepared by coating 96-well Corning tissue culture plates with FN at concentrations of 0, 0.1, 0.3, 0.6, 1.2, 2.4, 5.0, and 10.0 µg/ml for 2 h. Cells from confluent cultures were seeded at 6 10/well onto FN-coated plates and allowed to adhere for 90 min. After removal of unattached cells, adhesion was determined by the MTT assay. The effects of TGF- were determined by treating the cells for 48 h prior to the adhesion assay. A, no TGF-; B, 5 ng/ml TGF-. Each point is the average of two individual experiments determined from triplicate wells.



Since repression of autocrine TGF- activity down-regulated cell-surface integrin expression and cell adhesion to FN, it was of importance to determine whether the reduced cell adhesion to FN was mediated by loss of cell-surface expression of integrin . Therefore, blocking of adhesion to FN by monoclonal antibody against the subunit was performed (Fig. 6). Blocking with antibody showed 60 to 20% inhibition of adhesion to FN in FET Neo control cells at antibody dilutions of 1:50-1:500. In contrast, FET B cells showed very little dependence upon integrin for binding to FN as a 1:50 dilution of antibody resulted in only a 20% reduction of binding. Lower antibody concentrations did not give significant levels of inhibition of binding to FN. Antibodies to and subunits had little or no effect on binding to FN at 1:50 dilutions (Fig. 6A). Furthermore, blocking of adhesion to FN by antibody was also performed to determine whether restoration of cell adhesion to FN by treatment with exogenous TGF- was due to increased expression of cell-surface integrin (Fig. 6B). After treatment of FET B cells with 5 ng/ml TGF-, the levels of integrin -mediated adhesion to FN in FET B cells were similar to those in FET Neo cells at the same antibody dilution as shown in Fig. 6B. Therefore, it appears that repression of autocrine TGF- selectively reduces integrin -mediated adhesion to FN. Treatment with exogenous TGF- selectively restores the relative degree of integrin -mediated adhesion to FN in FET B cells.


Figure 6: Inhibition of adhesion of FET control () and FET TGF- antisense transfected (&cjs2110;) cells to FN by antibodies to integrin receptors. Ninety-six-well Corning tissue culture plates were coated with FN (10 µg/ml). Monoclonal antibodies to integrin subunits were added at dilutions of 1:50-1:500 and incubated for 30 min at 37 °C. A, no TGF-; B, 5 ng/ml TGF-. 1, no antibody; 2, antibody; 3, antibody; 4-6, antibody. Each point is the average of two individual experiments determined from triplicate wells.




DISCUSSION

It has been shown that exogenous TGF- is able to regulate the expression of integrins (13, 14, 15, 33) . These results indicated that the biological functions of TGF- and integrin-mediated cell-matrix interaction overlap in terms of regulation of cell growth, migration, and differentiation. However, demonstration that autocrine TGF- in a native cell line controls the steady-state expression of integrin receptors and their biological functions has not been reported.

A large bank of colon carcinoma cell lines with a broad spectrum of biological properties has been characterized in our laboratory (22) . We have previously shown that sensitivity to exogenous TGF- and TGF- is dependent upon the progression status of colon carcinoma cell lines (34) . Poorly tumorigenic cell lines that retain differentiated characteristics in tissue culture, such as basolateral polarity and transport function, respond to exogenous TGF- treatment with decreased proliferation, loss of anchorage independence, and increased expression of carcinoembryonic antigen and ECM proteins, including basement membrane components such as laminin and collagen IV in addition to FN (34, 35, 36) . Cell lines sensitive to exogenous TGF- were shown to have an autocrine negative TGF- function (21, 25, 29) . Interestingly, the ECM material produced by the cells with autocrine negative TGF- is able to induce a differentiation-like response in another colon carcinoma cell line, designated MOSER, whereas the ECM material from highly progressed colon carcinoma cell lines without autocrine negative TGF- is not able to induce this response (37) . Moreover, we recently showed that the expression of the subunit selectively blocks DNA synthesis in HT1080 fibrosarcoma cells (8) . Taken together, these lines of evidence suggest that autocrine TGF- might play an important role in controlling the biological properties through autocrine control of integrin and/or ECM protein expression and that the malignant progression seen in FET B cells might in part be due to loss of steady-state control of integrin function. Previously, it had been shown that tumor progression is associated with loss of TGF- responsiveness (17, 21, 34, 38) . Given the role of in the tumorigenicity of other cell types, it is possible that the steady-state levels of under autocrine TGF- control are important in suppressing the tumorigenic potential of FET cells.

The integrin subunit mRNA steady-state level and cell-surface integrin expression were down-regulated in FET TGF- antisense transfected cells compared with the control cells, whereas there was no change in the mRNA levels of integrin and subunits, indicating that the modulation of expression is relatively specific in this cell line. The decrease in cell-surface integrin expression and cell adhesion to FN by repression of autocrine TGF- in FET cells was reversed by treatment with exogenous TGF-, further confirming that the expression of integrin is under the control of autocrine TGF- regulation. A number of mechanisms for increased integrin expression resulting from TGF- treatment have been observed, including increased and subunit synthesis (14) and decreased decay (13) . We studied whether autocrine TGF- regulates integrin expression at a transcriptional level. The negative results indicate that autocrine TGF- does not appear to control integrin expression at the transcriptional level in FET cells. Thus, autocrine TGF- may function by increasing the stability of integrin mRNA, facilitating integrin protein incorporation into the cell membrane, and/or inhibiting the protein degradation.

In conclusion, our data show that the autocrine expression of TGF- has a role in modulating the steady-state expression of integrin and, as a result, controls integrin -mediated cell adhesion. Repression of autocrine TGF- activity in the FET colon carcinoma cell line resulted in diminished integrin expression and cell adhesion to FN. Exogenous TGF- treatment restored cell-surface integrin expression and integrin -mediated adhesion in the TGF- antisense transfected cells to the same level as in control cells. These results indicate that autocrine TGF- may affect cell growth and differentiation in part via modulation of the expression of ECM proteins and integrins.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA38173 and CA50457 (to M. G. B.) and Grant CA63480 (to L. S.) and by a grant from the American Cancer Society, Ohio Division (to L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Performed this work to fulfill partial requirements for a Ph.D. degree at the Medical College of Ohio.

Contributed equally to this work.

**
Performed this work to fulfill partial requirements for a Ph.D. degree in the Department of Pharmacology at the Baylor College of Medicine.

§§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Ohio, P. O. Box 10008, Toledo, OH 43699-0008. Tel.: 419-381-4131; Fax: 419-382-7395.

The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; TGF-, transforming growth factor ; CAT, chloramphenicol acetyltransferase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.


ACKNOWLEDGEMENTS

We thank Jenifer Zak for the skillful preparation of this manuscript.


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