Abnormal expression of the RI subunit of cyclic AMP-dependent protein kinase in Y-79 retinoblastoma cells

Exp.

Eye Res. (1989)

Abnormal Dependent SUSAN

48, 497-507

Expression of the RI Subunit of Cyclic Protein Kinase in Y-79 Retinoblastoma PAUL

GENTLEMAN,PT[

GERALD

RUSSELL,~ BRIAN J. CHADERt

A.

AMPCells

HEMMINGS~

AND

t Laboratory of Retinal Cell and Molecular Biology, $ Laboratory of Mechanisms of Ocular Disease National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, US, and SFriedrich Miescher Institut, Box 2543, CH 4002 Basel, Switzerland

(Received21 April

1988 and accepted in revised form

22 August 1988)

The cyclic AMP-dependent protein kinases of Y-79 retinoblastoma cells were analyzed by DEAE Sephacel column chromatography, SDS-polyacrylamide gel electrophoresis, and immunoblotting. Roth type I and type II protein kinases (characteristic of fetal and mature retina respectively) were observed. In addition, free RI (type I regulatory subunit) was present in Y-79 cells; three other mtinoblastoma cell lines also showed evidence of free RI in immunoblot analysis. Northern blot analysis of poly A+-enriched RNA from Y-79 cells showed a 45 kb RI transcript and a 6.0 kb RI1 transcript. The single 45 kb RI message is typical of normal retina, in contrast to other tissues and cell lines in which a 20 kb message predominates. Since RI expression appears to be post-transcriptionally inhibited in normal retina, it is suggested that free RI dimers in retinoblastoma is the result of loss of repression of translation of this transcript and that the free RI pool may contribute to the unregulated growth of these cells. Key words: retinoblastoma; protein kinase A; mRNA.

1. Introduction

Retinoblastoma, the most common pediatric ocular tumor, arises during the development of the retina as the result of the loss of both wild type allelesat a locus on the chromosomal region 13q141 (Murphree and Benedict, 1984; Cavanee et al., 1985; Friend et al., 1986) The retinoblastoma (Rb) gene is thought to have a ‘suppressor’ or growth-limiting function which results in a recessive pattern of expression of the mutant genes, as postulated by the two-hit theory of Knudsen (1971). The second hit, resulting in tumorigenesis, appears to be due to mitotic segregation events, such as chromosome loss with or without duplication of the mutant chromosome, mitotic recombination, and inactivation or microdeletion of the wild type allele (Gallie and Worton, 1986). To date, correlation of retinoblastoma with amplification or activation of known oncogenes as a primary event in tumorigenesis has not been found (Squire, Gallie and Phillips, 1985; Squire et al., 1986). Thus, although the Rb gene has been identified and sequenced(Friend et al., 1986; Lee et al., 1987a; Fung et al., 1987),the mechanismof growth regulation by this gene is not understood. Cyclic AMP is an important regulator of cellular metabolism, differentiation and gene expression (Krebs and Beavo, 1979; Lohmann and Walter, 1984; Jungmann, Constantinou, Kwast-Welfeld and Schweppe, 1986; Schlichter, Miller and Wicks, 1986). Cyclic AMP-dependent protein kinases, as the major receptors for cyclic AMP r To whom

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011&4835/89/040497+11 Li

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ET AL

in eucaryotic cells, are the mediators by which cyclic AMP exerts its effects on proliferation, differentiation, and transformation (Lohmann and Walter. 1984). The enzyme consists of a tetramer of t,wo cyclic AMP-binding regulatory subunits (R) and two catalytic subunits (C). Two isotypes, distinguished by diff’erent regulatory subunits (RI and RII), are recognized, and the ratio of the isotypes varies specifically with respect to cell type and developmental st,age. Thus. developmental or tissuespecific effects of cyclic AMP could be mediated by the isot’ype of protein kinase present. For example, in normal developing retina. the early fet’al kinase is a type I form. At the cessation of proliferation, the type 1 kinase decreases, and the type II kinase appears; in mature retina only type II kinase is found (Gentleman, Hemmings, Russell and Chader, in press). In the study presented here, we have investigated the expression of cyclic AMPdependent protein kinases in the Y-79 retinoblastoma cell line. The Y-79 cell line was derived from a tumor obtained from a child with hereditary retinoblastoma (Reid et al., 1974). These cells grow rapidly in suspension culture (i7 hr doubling time) and appear to be highly undifferentiated (Kyritsis, Tsokos, Triche and Chader, 1984; Jiang, Lim and Blodi, 1984), although some specific retinal proteins are expressed (Kyritsis, Wiggert, Lee and Chader, 1985). Studies of the cyclic AMP-dependent protein kinases in these cells may shed light on the role of these enzymes in both normal growth and malignant transformation in the retina.

2. Materials Sample

and Methods

preparation

Y-79 retinoblastoma cells were grown in RPM1 1640 medium containing 20 mM HEPES and 10 % heat-inactivated fetal calf serum. Cells were harvested by centrifugation, washed with phosphate-buffered saline and homogenized in a buffer containing 20 mM HEPES buffer (pH 7.4), 100 mM NaCl, 10 mM EDTA, 1 mM dithiothreitol. 50 ,ug ml-’ leupeptin, and 61 mM phenylmethylsulfonyl fluoride. Cytosolic fractions were obtained by centrifugation of the homogenate at 27000 x g for 20 min. The supernatant fractions were adjusted to a protein concentration of 1-5 mg ml-’ and stored at -2OOC. Frozen pelleted cells of Y-83, Y-83NH, and W-24 were the kind gift of Dr D. A. Albert, Massachusetts Eye and Ear Hospital, Boston, MA. Human retinal tissue was obtained from cornea1 donors with no known ocular pathologies. Eyes were enucleated within 3 hr of death, the corneas removed, and the posterior poles maintained at 4°C until removal of lens and retina (12-24 hr post mortem). Fetal retina was obtained from eyes of 12-24 week fetuses enucleated within 1 hr of the operation and maintained at 4°C no more than 8 hr before removal of the retinas. Cytosolic fractions were prepared as for the retinoblastoma cells. Cyclic

AMP

binding

asmy

Cyclic AMP binding was determined by the method of Gilman (1970). The incubation medium contained 50 mM Na acetate buffer (pH 40), l&60 nM [3H]-cyclic AMP (specific activity = 31.6 mCi ,umol-l), and 61 mg ml-’ protein kinase inhibitor protein in a final volume of 50 ~1. AMP-dependent protein kinase assay Protein kinase activity was measured by a modification of the method of Roskoski (1983). The reaction medium contained 50 miw imidazole HCl buffer (pH 7.2). 10 mm MgCl,, 61 mM isobutylmethylxanthine, @lo? (w :v) bovine serum albumin, 1 mM EGTA, 1 mM dithiothreitol, 100 ,uM $‘P]ATP (specific activity = 20 mCi mmol-i), 65 mM Kemptide (Sigma, St’ Louis, MO), 004-O 1 mg ml-’ enzyme protein, and 10 ,uM cyclic AMP where noted in a final volume of 50 ,uI. After a 5 min incubation at 37”C, the reaction was terminated by application of 40 ,~l aliquots to Whatman P81 filter paper squares, which were dropped into 5 mM Cyclic

PROTEIN

KINASE

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499

phosphoric acid. The filter papers were then rinsed three times in acid and counted. Reaction blanks of 150-200 cpm were routinely obtained. Cyclic AMP-dependent protein kinase activity was linear with time and protein concentration under these assay conditions. DEAE Sephacel cohmn chromatography A DEAE Sephacel column (@9x 30 cm, volume = 19 ml) was equilibrated with 20 mM HEPES (pH ‘7.4) containing 50 mM NaCl, 1 mM EGTA. 10 pg ml-’ leupeptin, and 1 mM dithiothreitol. The cytosolic fraction of Y-79 cells, dialyzed against t,he column equilibration buffer. was applied and the column washed with two volumes of equilibration buffer with a flow rate of 6 ml hr-‘. The column was then eluted with a 50-400 mM NaCl gradient in four column volumes. Fractions were dialyzed against NaCl-free buffer and assayed for cyclic AMP binding and cyclic AMP-dependent protein kinase activities. The gradient was determined by measurement of conductivity every fifth fract’ion. SDS-polyacrylamidp gel electrophoresis and immunoblot SDS-polyacrylamide gels were run according to the method of Laemmli (1970) with a 10 % resolving gel and a 4”/0 stacking gel. Pre-stained molecular weight markers (BRL. Gaithersburg, MD) were used as standards. The proteins were transferred by electroblot to nitrocellulose paper according to the procedure of Towbin. Staehelin and Gorden (1979). to bovine cardiac and skeletal kinase subunits Affinity-purified rabbit antibodies (Hemmings, 1986) and gold-coupled goat anti-rabbit antibodies (Boehringer-Mannheim. Indianapolis. IN) were used to visualize the proteins on the blots. Protein concentration Protein concentrations were determined by the dye-binding using fatty acid-free bovine serum albumin as the standard.

method

of Bradford

(1976),

RI an.d RII cDNA probes Clones for RI and RI1 were isolated from a Agt-11 cDNA library of LLC-PK, cells as previously described (Nowak, Seipel, Schwarz, Jans and Hemmings, 1987; Hemmings, Schwarz, Adavani and Jans, 1986). The cDNA probes for RI and RI1 were made from /\RI 15 and hRI1 17 clones, labeled by the random priming method (Feinberg and Vogelstein, 1983). Northern

blot analysis RNA was isolated on cesium chloride gradients, and poly A+-enriched RNA was isolated by application in buffer containing LiCl and batch elution from oligo-dT cellulose 1982). The RNA was separated on 1% aga(Maniatis, Fritsch and Sambrook, rose-formaldehyde gels and transferred to Genescreen (NEN, Boston, MA) by capillary blotting (Maniatis et al., 1982). Prehybridization, hybridization and washing of the blot was done according to the manufacturer’s instructions. Total

3. Results Comparison

of protein

kinase activities

in retina

and retinoblastoma

Cyclic AMP-dependent protein kinase activity in Y-79 cell cytosolic fractions was lower than in mature retina but not significantly different from that in fetal retina (Table I). However, there were significantly fewer cyclic AMP binding sites per mg extract protein in the Y-79 cells than in fetal retina, as shown by the Scatchard analysis of the binding (Fig. 1). This difference may be due to a 70 kD cyclic AMP binding unique to fetal retina (Gentleman et al., in press). DEAE

Sephacel

chromatography

of Y-79 cell protein

kinases

Cyclic AMP-dependent protein kinase isotypes are characterized on the basis of their elution from DEAE exchange columns, the type I form eluting between 50 and 100 mM NaCl and the type II eluting between 200 and 300 mM NaCl (Tao, 1974). As Ii-2

500

S. GENTLEMAN

ET

TABLE

AL.

I

Cyclic AMP-dependent protein kinase activity in adult and fetal human retina and I’79 retinoblastoma cells. Cytosolic fractions were prepared and assayed as described Protein kinase activity (pm01 mg-’ min-‘)*s.E.M Tissue

-Cyclic

Adult retina Fetal retina (12-15 Y-79 retinoblastoma

AMP

169k3.5 169k71 98fl5

weeks)

+Cyclic

AMP

N

608_f

92

3

325* 103+_

154* 31*

5 4

N = Number of samples assayed. All assays done in duplicate. * P < @05, Duncan’s test.

1 (a)

IO

0

(b)

20 c3H]

cyclic

0

40 AMP

(nM)

FIG. 1. [3H]-Cyclic AMP binding (triangles), 12-15 week fetal retina of (a).

20

40

60

80

Bound

in retina and retinoblastoma. (a) Cytosolic fractions of adult retina (closed circles) and Y-79 cells (open circles). (b) Scatchard analysis

shown in Fig. 2, chromatography of the Y-79 cell extract gave a small peak of type I activity (fractions 2CL25) and a large, somewhat heterogeneous peak of type II activity (fractions 28-38). In a series of four such columns, the type I peak varied quantitatively, contributing between 10 and 40% of the total kinase activity; the activity in the type II peaks varied two-fold among these experiments (data not shown). There was also a peak of cyclic AMP binding activity which did not correlate well with the kinase activity that eluted on the leading shoulder of the type II holoenzyme peak (Fig. 2). This is the expected elution position for RI dimer (Tao, 1974) and probably represents an excess regulatory subunit, as no free catalytic activity was detected in the run-through volume of the column (fractions 5-10).

PROTEIN

KIBASE

IN

RETINOULASTOMA

Fraction

number

FIG. 2. DEAE Sephacel chromatography of Y-79 cytosolic fraction. cyclic AMP binding; (e-O-@) protein kinase activity with 10,~~ kinase activity without cyclic AMP: (---) N&l concentration.

(- ) protein; cyclic AMP;

(n---A---A) (O-0-0)

[3H]protein

(b)

15-

IO -

5-

o-

26 Fraction

FIG. 3. (a) DEAE Sephacel chromatography AMP bound ; (O-0-0) cyclic AMP-dependent from (a) RI. RI1 and C! subunits from normal

number

20 30 32 Fraction

of Y-79 cell cytosolic fraction. (@-o-0) protein kinase activity. (b) Immunoblots human fetal retina are indicated.

[3H]-cyclic of fractions

S. (:ENTLEMAS

502

ET

AL.

28s

18s

(a)

(b)

FIG. 4. Northern

blot analysis of poly X+-enriched RNA from Y-79 cells. The blot (duplicate Y-79 RNA samples applied) was first hybridized with the RI probe (a), and then. without stripping the RI probe from the blot, the blot was re-hybridized with the RI1 probe (b). Hybridization was carried out at 60°C and blots were washed 3 times at the same temperature using 2 x SSC (150 mM NaCl with 15 mM Na citrate) containing 0.1 %I SDS.

To verify the presence of the RI subunit on the shoulder of the type II holoenzyme peak, fractions 26, 28, 30, and 32 from the DEAE column shown in Fig. 3(a) were concentrated by ultrafiltration and subjected to SDS-polyacrylamide gel electrophoresis and electroblotted (Fig. 3(b)). A n intense RI band is seen in fraction 28 on the leading shoulder of the type-II holoenzyme peak, whereas both RI1 and C immunoreactivity showed a maximum intensity in fraction 30. Taken with the lack of free catalytic activity in the wash through volume of this column, these data suggest that the RI subunit in Y-79 cells exists as the free dimer.

Northern

blot analysis

of RI and RII ,in Y-79

cells

Northern blots of poly A+-enriched RNA from size for each subunit (Fig. 4). The RI mRNA normal retinal RNA (Gentleman et al., in press). to that reported for several cell lines and tissues 1987).

Y-79 cells showed a single transcript was 45 kb, exactly as that seen in The RI1 message was 60 kb, similar (Hemmings et al., 1986 ; Scott et al.,

PROTEIX

KIXASE

IN RETINOBLASTOMA TABLE

,503

II

Cyclic AMP-dependent protein kinase activity and [3H]-cycZic AMP binding in, retinoblastoma cell lines. Cytosolic fractions were prepared from frozen cells and assayed in duplicate for the binding assay and in triplicate for the kinase assay Protein kinase activity (pm01 rnp-’ min-‘)

Y-79t Y-83$ Y-83NH: W-24$

116 72 102 16:!

.T&S.E.M.

105+

1“H J-C ‘yclic

AMP bound (pmol mg-‘)

-

365 210 444 404 334*51

19

t Reid et al. (1974); 3: Potluri et al. (19%): § isolated by T. W. Rery. Wills Eye Research Institute, Philadelphia, PA.

(b)

(a)

C

43K

SI 2 34 MW FIG. 5. Immunoblots of cytosolic fractions from retinoblastoma cell lines after SDS polyacrylemide gel electrophoresis. (1) Y-79; (2) Y-83; (3) Y-83NH; (4) W-24. (a) Anti-RI. (b) Anti-RII. 2@30 pg protein were applied to each well. MW, BRL prestained molecular weight markers: S, bovine protein RI and RI1 subunits. The standard in (b) was overloaded. MW

Comparison

I

2

3

4

S

of Y-79 cells with other retinoblastoma

cells

Cytosolic fractions were prepared from several other retinoblastoma cell lines for analysis of cyclic AMP binding and cyclic AMP-dependent protein kinase activity and isotypes. As shown in Table II, the activities fell within the range obtained previously with Y-79 cells and fetal retina (see Table I). Aliquots of the cytosolic fractions were then subjected to SDS-polyacrylamide electrophoresis and electroblotted. In all cell lines, a 49 kD band of RI immunoreactivity was observed ; two to three smaller bands of RI immunoreactivity, which were apparent proteolytic fragments, were also detected (Fig. 5(a)). A single band of RI1 immunoreactivity with an Mr of about 50 kD was seen in all cell lines (Fig. 5(b)).

504

S. GENTLEMAN

ET

AI,

4oc

300

67 b -5 =u) s B & 2 1

200

100

Days in culture

FIG. 6. The effect of cyclic AMP analogs on the proliferation of Y-79 cells in suspension culture. Six low density cultures (3 x lo4 cells in 20 ml of RPM1 1640 containing 10% fetal calf serum) were established, two containing no additions, two containing 1OOpM dibutyryl cyclic AMP, and two containing 100 pM 8-bromocyclic AMP. Cell counts of 3 ml aliquots from each culture were done daily for four days, and 25 ml of additional medium (containing cyclic AMP analogues where appropriate) was added to all cultures on day 2. (-O-O-) Control cultures; (-O-O-) cultures containing 100 FM dibutyryl cyclic AMP ; (-A-A-) cultures containing 109 FM 8~bromocyclic AMP. Effect of cyclic

AMP

analogues

on

Y-79

cell

growth

Y-79 cells were seededinto culture media containing 100 ,uM dibutyryl cyclic AMP or 8-bromocyclic AMP for comparison with untreated cultures. At 1 day intervals, all cultures were sampled and counted with a Coulter counter. As shown in Fig. 6, there were no differences in cell proliferation rates in the three culture conditions over a four-day period. Therefore, in short term experiments, Y-79 cells appear to be resistent to the growth inhibitory effects of cyclic nucleotides. 4. Discussion In normal human retina, cyclic AMP-dependent protein kinase changes from the type I enzyme to the type II enzyme in the mid-period of fetal development after the

PROTEIN

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505

cessation of mitotic activity (Gentleman et al., in press). In the Y-79 cells, both isotypes were seen. The data from the other retinoblastoma cell lines suggest that Y79 cells are not unique in expressing two isotypes. Moreover, all lines showed smaller RI immunoreactive bands, suggesting proteolytic sensitivity of the RI subunit in retinoblastoma cells. Expression of RI in normal fetal retinal tissue does not show such smaller bands (Gentleman et al., in press). Although the ratio of R to C sub-units is 1: 1 in normal tissues (Corbin, Sugden, Lincoln and Keely, 1977 ; Hofmann, Bechtel and Krebs, 1977), an excess of regulatory subunits has been reported in several transformed cell lines (Singh, Roth, Gottesman and Pasten, 1981; Steinberg and Agard, 1981; Schwartz and Rubin, 1983, Rogers, Narindrasorasak, Cates and Sanwal, 1985 ; Botterell, Jans and Hemmings, 1987). The Y-79 cell line showed RI immunoreactivity eluting in the free dimer position on the DEAE column, but no free catalytic activity in the run-through volume, suggesting imbalance in the R:C ratio in these cells. Moreover, gel electrophoresis and blotting of the cytosolic fractions of Y-79 cells and other retinoblastoma cell lines showed a series of smaller RI immunoreactive bands which are likely to be proteolytic fragments of RI. Steinberg and Agard (1981) h ave shown that free RI is subject to proteolytic degradation at ten times the rate of this subunit in the holoenzyme. In contrast, no smaller bands suggestive of degradation were seen in the RI1 blots of these samples, and no indication of free RI1 subunit was found in DEAE column analysis of the Y-79 cell line. A consequence of malignant transformation in retinoblastoma, therefore, may be an uncoordinated synthesis of RI subunit in these cells. Unlike many cell types and cultured cell lines (Friedman, 1982), Y-79 cell proliferation was not inhibited by cyclic AMP analogs. Perhaps the pool of free RI dimer plays a role in this resistence to the growth inhibitory effects of cyclic AMP contributing to the unregulated growth of the tumor. Northern blot analysis of the regulatory subunit transcripts of Y-79 cells showed a single transcript for each isoform. The 45 kb RI message is of particular interest because several other cell lines and tissues expressing the RI isoform show a 2.0 kb message in addition to the larger transcript (Nowak et al., 1987). In contrast, in retina, only the 4.5 kb transcript is found, in both fetal and adult retina, even though the protein is only expressed in fetal retina (Gentleman et al., in press). Thus, expression of RI in retina appears to be post-transcriptionally regulated. One may speculate that, in retinoblastoma, the suppression of translation of the RI transcript has been lost, leading to the synthesis of RI in excess of C. Since Lee et al. (198713) have recently shown that the Rb gene product has nucleic acid binding properties, it is possible that one of the Rb gene repressor functions could be regulation of translation of the 4-5 kb RI transcript. The loss of this response and the overof the RI subunit may explain the unregulated growth of the expression retinoblastoma cells. Studies to identify proteins other than C that are affected by RI dimer are presently underway in this laboratory.

ACKNOWLEDGMENTS Human retinal tissue was obtained through the National Disease Research Interchange, Philadelphia, PA.

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ET

AL.

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