Molecular and Cellular Biochemistry 112: 73-79, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Expression of c-myc protooncogene in rat lens cells during development, maturation and reversal of galactose cataracts Yi W e n , 1 Songtao Shu, 1 Nalin J. Unakar 2 and Isaac B e k h o r 1 1Laboratory for Molecular Genetics, Doheny Eye Institute and School of Dentistry, University of Southern California, 1355 San Pablo St., Los Angeles, California 90033, USA and 2Department of Biological Sciences, Oakland University, Rochester, Michigan 48309, USA Received 16 September 1991; accepted 29 January 1992

Abstract It is well established that normal patterns of epithelial cell proliferation and metabolism, and of fiber cell differentiation and maturation are essential for the maintenance of transparency in the ocular lens. Several factors, including exposure to high levels of sugars, have been known to result in the compromise of lens transparency. For example, initiation of lens cell damage by galactose induces lens epithelial cells to proliferate. Elevated levels of c-myc m_0,2qA have usually been correlated with rapid cell growth and increased entry of cells into the S phase. Therefore, changes in c-myc mRNA levels may provide an early indication of the stimulation of lens epithelial cells to proliferate and differentiate, which has been postulated to be an early and important event in response to lens cell injury by galactose. By Northern blot hybridization analysis we quantitated c-myc mRNA levels in the lens capsule epithelia of rats (1) exposed to galactose, and (2) undergoing a partial recovery from the galactose-induced cell damage. At the onset of lens cell damage, we find c-myc mRNA to elevate to 6-fold by 24 hr, and by 48 hr decreases to about 3-fold the normal levels. During recovery, c-myc mRNA continues to be expressed at high levels approaching a 10-fold increase by day 12, then decreasing to levels of about 8-fold the control by day 30. The 24 h transitory elevation in c-myc mRNA in lens epithelial cells is in accord with our previous observations on the 24 h increase in MP26, ycrystallin and aldose reductase mRNAs following a high influx of galactose. Therefore, the elevation in c-myc mRNA as well suggest that galactose appears to cause lens cells to undergo an early transitory period of gene induction following the exposure of lens cells to galactose. (Mol Cell Biochem 112: 73-79, 1992)

Key words." c-myc mRNA, y-actin, lens epithelial cells, galactose cataracts, Northern blot hybridization, epithelial cell proliferation, reversed galactose cataracts

Introduction Elevated levels of c-myc mRNA are usually correlated with rapid cell growth and increased entry of cells into S

phase [1-3]. In several instances elevation of c-myc mRNA levels has been observed in differentiating cells

Address for offp~nts." I. Bekhor, Gerontology Center, University of Southern California, 3715 McClintock Avenue, Los Angeles, CA 90089-0191,

USA

74 [4~5], including lens cells [7, 8], and other mammalian cells in response to a variety of mitogenic substances such as growth factors [3, 9], hormones [10] and cyclic AMP [11]. The course of induction of mRNA in a stimulated tissue, such as regenerating fiver [12], follows a pattern characteristic to cultured mitogen-induced fibroblasts: a transient increase in the level of the c-los mRNA [13], followed by an increase in c-myc mRNA [14], and Ha-ras mRNA [15]. In particular, accumulation of c-myc mRNA was associated with DNA replication in a tissue undergoing regeneration, such as the liver [12]. To date, a body of evidence suggests that c-myc may be an important growth regulator whose expression in normal cells may be influenced by extracellular signals that modulate proliferation. Similar conclusions were derived from various studies on lens epithelial cells. A transient increase in c-myc mRNA was found to occur in lens epithelial cells stimulated to differentiate in culture [7, 8]. This change in the levels of c-myc mRNA may be a general feature of cell differentiation, thus the c-myc protein may play a distinct role in division and differentiation. On the other hand, decreased mRNA levels for c-myc were reported in cells undergoing terminal differentiation [16]. The bulk of the lens is made up of transparent fiber cells, while a monolayer of epithelial cells covers the anterior surface of the fiber cell mass. In vivo, induction of lens cell injury induces the monolayered lens epithelial cells to proliferate and become multilayered. This proliferative activity of the epithelial cells is initiated within the first 24 hr following the in vivo exposure of the lens to galactose [17, 18]. In contrast, when galactose is administered to lens epithelial cells in culture, these cells do not appear to be stimulated to proliferate but rather form vacuoles and appear to decrease in number [19, 20]. Therefore distinct mechanisms controlling the in vivo vs the in vitro response of the lens epithelial cells to galactose are implied. Galactose is not known to be an mitogenic agent but it is a well recognized cataractogenic agent leading to fiber cell disintegration [21]. In the present study we quantitated the levels of c-myc mRNA found in lens capsule epithelial cells isolated from rats exposed to high concentrations of galactose, followed by a similar study on lens undergoing recovery from the galactose-induced cell damage.

Experimental procedures Galactosemic rat lenses The animals used for this investigation were 50 g female Sprague Dawley rats (4 weeks of age; King Animals, Inc., Madison, MI) fed a diet containing 50% ground Purina Rat Chow and 50% D-galactose for up to 20 days following a 24 h fasting period. Induction of recovery was carried out by replacing the 50% galactose diet with a diet of Purina Rat Chow alone on day 20. Then the rats were maintained on the latter diet for an additional period of up to 30 days. At pre-designated times as indicated in the Figures below, lenses were enucleated then stored at - 70° C until used for decapsulation and isolation of RNA. Animal use was in accordance with guidelines in the 'Declaration of Helsinki' and 'The Guiding Principles in the Care and Use of Animals' (DHEW publication, NIH 86-23).

Isolation of RNA from lens capsule Lenses were decapsulated and the capsules were processed for the isolation of capsule epithelial cell RNA as previously described [22[. Briefly, a single lens capsule from each group was hand-homogenized at room temperature into 100 IxL of 0.5% SDS-0.025 M Na2EDTA (pH 8.0)-0.075 M NaC1 and 0.01M Tris-HC1 (pH 8.0)saturated redistilled phenol (pH 7.5), to which 25 tzg (1 txL) of yeast tRNA was added as a cartier. The aqueous phase was separated by centrifugation in a clinical centrifuge at 4000 r/min for 5 min at room temperature. The aqueous phase was extracted a second time with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) for 5 min at room temperature, and recovered by centrifugation as above. The final aqueous phase was extracted three times with ether then made 0.3M in Na-acetate (pH 6.0), to which 2.5 volumes of - 20° C ethanol was added to precipitate the nucleic acids overnight at - 20° C. The RNA/DNA precipitate was collected by centrifugation at maximum speed in a microcentrifuge at 4° C for 10 min. The pellet was dissolved in about 5txL of 0.1% diethylpyrocarbonate (DEPC)treated sterile distilled water. The RNA solution was stored at - 70° C for further use.

75

Quantitation of poly(A + )-mRNA for electrophoresis The procedure used is essentially that of Fornace and Mitchell [23] utilizing poly(dT) probe to estimate quantity of m R N A based on the poly(A+)-content in our R N A sample relative to a standard poly(A+) mRNA. The procedure was reviewed in details by Farrell [24]. In brief, poly(A) (Sigma, St. Louis, MO) was used as a template for synthesis of [32p]-poly(dT) with M-MLV reverse transcriptase in a reaction volume of 100~L containing: 100txCi c~[32p]-dTYP (3000 Ci/mmol; DuPont/NEN, Boston, MA), 25 txg poly(A), 2 mM dithiothreitol, 5 0 m M Tris-HC1 (pH 8.3), 40mM KCI, 20 mM MgC12, 50 U RNasin (Promega, Madison, WI), 1 mM dTI'P, 5 txg oligo dT12_~8(Sigma), 1000 U M-MLV reverse transcriptase (BRL, Gaithersburg, MD), and incubated at 37 ° C for 1.5 hr. The reaction was terminated by the addition of 100 mM N a O H and 10 mM EDTA, and incubated at 65°C for 30 min, neutralized with 100 mM Tris-HC1 (pH 7.5), and the labeled probe was separated from unincorporated nucleotides by spun column chromatography through Sephadex G-25. Following spotting on nitrocellulose filters, known volumes (up to 1 txL) of an unknown R N A quantity vs a known quantity of from 0.2 to 1/xg per/xL of poly(A+) globin m R N A (BRL), filters were pre-hybridized as described [25], then hybridized at 42 ° C for 4 hr, processed for cpm hybridized as described [25], exposed for 2 days to Kodak XAR-5 film at - 70 ° C, and quantitated by laser scanning densitometry [26] for estimating #g poly(A+) m R N A present per/xL of sample relative to the standard poly(A+) globin mRNA. Yeast tRNA of up to 5txg was also spotted and used as a negative control.

Preparation of antisense c-myc RNA probe c-Myc R N A probe was prepared by subcloning of the feline exon II 1.0 kb Sst I fragment of ~. clone CM-2 of Soe and Roy-Burman [27] into the transcription vector pBS (Stratagene, La Jolla, CA) containing T3 and T7 transcriptional promoters in opposite polarities into the coding region of the lacZ gene of pUC19 [28]. Sense [35S]-UTP labeled R N A was generated by transcription of the recombinant pBS-cmycII with T3 R N A polymerase (Promega) following its restriction with Eco R1 (BRL). Antisense [35S]-UTP labeled R N A was generated by transcription of pBS-cmycII with T7 R N A polymerase (Promega) following its restriction with Hind III

Fig.1. Northern blot hybridizationanalysisof c-mycmRNA levelsin lens capsule epithelial cells undergoinginductionof lens cell damage by dietary galactose. Lanes: 1, control, 28 day old rat lens; 2, 6hr on galactose; 3, 12hr; 4, 24 hr; 5, 48 hr; 6, 72 hr; 7, 4 days; 8, 8 days; 9, 12 days; 10, 16 days; 1l, 20 days. One p~gof poly(A+)mRNA, quantitated relative to poly(A+) globinmRNA as described in Methods,was electrophoresed on each lane (we estimate this to be equivalent to about 15p~g of total lens capsule RNA based on measurements of concentrationsof total RNA by ethidiumbromide, ref. 36). Each lane represents a separate pool of lens capsules, c-myc RNA probe was labeled with [35S]-UTP, and y-actin with [32p]-UTP as described in Methods. The relativesize of c-mycmRNA is 2.2 Kb as determinedin comparison to a RNA ladder (BRL), analogous in size to the one reported for other ocularcellsin culture [7, 39]. The lowabundanceof c-myc mRNA in lens necessitatesthe poolingof about 5~6 capsules for the measurementsdescribed in this figure.

(BRL). The [35S]-labeled transcripts were purified as described [29]. Northern blot quantitation For Northern blot hybridization analysis, a solution of R N A containing an equivalent number of hybridized poly(A+) mRNA, based on the cpm of poly(dT) hybridized as described above, and estimated to constitute about 1 txg of poly(A+) m R N A (about 15 Ixg total lens capsule RNA), was transferred to a 0.6 mL microfuge tube, and the RNA, denatured by glyoxalation in dimethyl-sulfoxide (DMSO) as described by Thomas [30], was electrophoresed on a 1% agarose gel in 0.01 M NaH2PO4 (pH 7.0). The gel was run submerged in buffer at 3-4 V/cm. At the end of the run the gel was stained for 15 min with 5.0/xg ethidium bromide per mL of 0.5 M NH4-acetate, then destained for 15 min in 0.5 M NH4-acetate, visualized by exposure to UV and photographed. The R N A was later transferred to nitrocellulose paper using the Hoefer Vacuum Blotter (Hoefer Scientific, San Francisco, CA). Transfer was done in 10 X SSC (0.15 NaC1, 0.015 Na-Citrate) and was complete in about 2 hr at room temperature. The transferred RNA was crosslinked to the nitrocellulose paper by

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analysis[26]. Each pointrepresents an averageof two separatedeterminationson separatepoolsoflenscapsules.Foldincreaseis determined by the intensityof each band relative to the controllevel (lane 1, Fig. 1) whichis set at 1. The transient elevationin c-mycmRNA is found to occur by 24 hr on galactose. exposure to ultraviolet radiation in a Stratalinker UV Crosslinker (Stratagene). The nitrocellulose RNA blots were processed for pre-hybridization and hybridization as follows: The pre-hybridization solution contained per 1.0mL: 500/xL formamide (deionized and 2 x recrystallized), 60/zL 5M NaC1, 201zL 1M Tris-HCl (pH 7.5), 50/xL 100mM EDTA (pH 7.5), 20/zL 50 X Denhardt's solution (1% polyvinylpyrrolidine, 1% Ficoll, 1% RNasefree bovine serum albumin), 0.1% sodium dodecylsulfate (SDS), 1/xL (20 mg/mL) RNase-free BSA, 20/xL (10mg/mL) denatured-sheared salmon sperm DNA, and 10/zL (25mg/mL) heat-denatured yeast transfer RNA. The final volume was adjusted to 1.0mL with DEPC-treated sterile distilled water. Pre-hybridization was carried out at 42°C for 2-4 hr in a heat-sealed hybridization bags. Following pre-hybridization, the solution was replaced with a fresh pre-hybridization solution containing antisense [35S]-RNA transcript at 4 X 106 cpm per mL (heat-denatured at 80°C for 5 min) and 1/5 volume of a freshly prepared 50% polyethylene glycol (PEG, 8000 d; Sigma) solution (in DEPC-treated water). The bag was resealed and hybridization was done at 60° C for a period of 24 hr. Following hybridization the filters were washed two times for 30 min each at room temperature in 1 X SSC-0.1% SDS, then washed two times for 30 min each at 65°C in 0.1 X SSC-0.1% SDS. Finally, the filters were exposed for 3 days to Kodak XAR-5 film at - 70° C. Following autoradiography the film was scanned in a two-dimensional soft laser scanning densitometer and quantitated relative to standard cpm of [35S]-labeled RNA [26]. Quanti-

Fig. 3. Northern blot hybridization analysis of c-myc mRNA levels in lens undergoing recovery from the galactose-induced cell injury. Recovery is accomplished by switching from the 50% galactose diet to a diet containing Purina Rat Chow alone by day 20 on galactose, at an age of 48 days. Lanes: 1, 28 day control; 2, 48 day control, 3, 1 day of recovery, equivalent to a 49 day old rat; 4, 6 day recovery; 5, 12 day recovery; 6, 17 day recovery, 7, 24 day recovery; 8, 30 day recovery.

tation values were further corrected for background hybridization levels by subtracting cpm hybridized of the same RNA population with sense [35S]-labeled riboprobes. The laser scan data was plotted by the Sigma Plot software (Jandel, Corte Madera, CA). Filters were subsequently stripped of initial probes and rehybridized with [32p]-labeled y-actin RNA-probes, transcribed from ¥-actin cDNA [31] subcloned into the transcription vector pSP64 (Promega, Madison, WI), for confirmation of quantity of cellular RNA electrophoresed per lane on agarose gels.

Results Figure 1 shows the results of c-myc mRNA Northern blot hybridization analysis on RNA isolated from lens epithelial cells of control vs galactose fed rats, along with the analysis of the quantity of y-actin mRNA present in the same RNA samples. The data from Fig, 1 are quantitated in Fig. 2. Figure 2 shows fold increase occurring in both c-myc and y-actin mRNAs in response to a high influx of galactose into the lens (up to 1000 nmoles/lens in a 24 h period, determined separately). Fold increase in mRNA was determined from the total intensity of a specific band as calculated by computer driven laser densitometry [26]. The results show that the elevation in c-myc mRNA levels begins by 6-12 hr following induction of cell damage by galactose. By 24 hr we find an increase by about 6-fold over the control level (time 0 in Fig. 2), That elevation is transient, and a drop in the level of c-myc mRNA begins by 48hr on galactose. That level appears to fluctuate slightly and remains at about 3-fold the 0 time level throughout the remainder of the time of exposure to galactose. On the other hand, y-actin mRNA does not

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Fig. 4. Quantitation of data from Fig. 3 as done by laser scan densitometry. Each point represents an average from two determinations on separate pools of lens capsules. A significant increase in c-myc mRNA levels is evident by day 12 of recovery. Filled square symbol at time 0 specifies the level of c-myc mRNA in normal lens at the age of 48 days. The zero time level, shown in filled circle, marks the c-myc mRNA level on day 20 on galactose (from Fig. 2). The 'steady state' level of c-myc mRNA is approached by day 17 of recovery; it is about 8-fold the normal level at time 0, and about 2-fold the level observed to occur in the presence of galactose at day 20 (Fig. 2).

appear to change significantly (Fig. 2, dotted line). The data of Figs. 1-2 establish that the early response of the lens epithelial cells to galactose may also be manifested by an elevation in c-myc m R N A by 24 hr, followed by a decline to a steady state level that appears to remain constant for the duration of the time of exposure to galactose. During that time period epithelial cells proliferate, as has been repeatedly shown by histological studies [32, 17]. Previous studies have shown that upon removal of the cataractogenic agent galactose from the diet, lens cell damage recede from the cortical and nuclear regions to the nuclear region of the lens [33]. Therefore, normal fiber cell proliferation and recovery appear to begin immediately following reduction in intracellular polyoN. We then quantitated the changes that may occur in c-myc m R N A levels as a result of induction of recovery from the galactose-induced cell damage. Figure 3 shows the Northern blot hybridization analysis on cmyc m R N A levels in epithelial cells at various times following removal of galactose from the diet. The data is quantitated in Fig. 4, showing that c-myc mRNA concentrations are elevated to higher levels (about 10fold the 0 time measure) than what was found to occur in lens immediately following dietary-induction of accumulation of intracellular polyols. The control levels for c-myc mRNAs in lens epithelial cells are shown both for the 28 day old rats in Fig. 2 at time 0, and for the 48 day

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Fig. 5. Abundance of c-myc mRNA in rat lens relative to total ctA-crystallin mRNA. RNA was isolated from undecapsulated lens as described [37], then processed for Northern blot hybridization analysis as described in Methods. Two separate determinations were done as shown in the figure. The hybrid bands were quantitated by laser scan densitometry, and plotted by the Harvard Graphics software. Calculation of the data shows that c-myc mRNA is present in control rat lens at 0.94% relative to the ctA-crystallin mRNA.

old rats in Fig. 4 at time 0 (symbol, solid square). Both values appear to be low and of the same magnitude, signifying a low steady state synthesis of c-myc mRNA occurring in lens epithelial cells, which are known to carry on a slow rate of continuous differentiation throughout the life of the lens [34]. The relative abundance of c-myc mRNA found in the lens is determined in Fig. 5. Calculation of the data shows that c-myc mRNA concentration in normal 28 day old lenses constitutes about 1% of the total ctAcrystallin mRNA found in that lens. ctA-crystallin mRNA is present at highest concentrations in the lens [37], and it is used by us as a primary level for measurement of relative abundance of other mRNAs in the lens.

Discussion The results in Figs. 1 and 2 demonstrate that c-myc mRNA elevation in lens epithelial cells is an early event occurring during induction of cell damage by galactose. It has been suggested, the timing of c-myc expression may be indicative of cell differentiation [35], and in our case it may also reflect active cell proliferation. Since lens epithelial cells begin to proliferate within 24hr following the introduction of galactose, the 24 h elevation in c-myc mRNA may be an early indication of increased entry of cells into S phase [1, 3]. However, we

78 recently reported that during this 24h period the mRNA for the main intrinsic fiber cell membrane protein, MP26, was elevated to about 3-fold the control, level [36]. This increase is being associated with enhanced lens epithelial cell differentiation to fiber cells, as is evidenced from our in situ hybridization studies [29]. Therefore, it is being proposed that at the 24 h period both epithelial cell proliferation and differentiation are significantly enhanced in response to the effect of galactose on lens cell integrity. Although elevation in c-myc mRNA has always been correlated with mitogeninduced cell growth [35], this is the first case where an agent that is known to bring on cell death in the lens [19, 21] is found to induce elevation in c-myc mRNA. The data favor the proposal that the cataractogenic agent galactose causes damaged lens cells (the fiber cells) to attempt recovery, i.e. by invoking activation of mechanisms promoting proliferation and differentiation of their progenitor cells (the epithelial cells located at the lens anterior capsule). The latter observation brings forth a very important concept suggesting that galactose-cataractogenesis is initiated in the lens epithelial cells. The decrease in c-myc mRNA by 48 hr relative to the 24 h levels on galactose coincides with the decrease in other lens mRNAs at this time period, as we recently reported [36, 37]. However, the continued high level of expression of c-myc mRNA between 48 hr and 20 days on galactose relative to the controls may be reflecting continued epithelial cell proliferation occurring during that period [17, 18]. Figures 3 and 4, on c-myc rnRNA elevation in lens epithelial cells during the recovery period, show a similar yet a slow transient increase in c-myc mRNA occurring during that time period. The elevation in c-myc mRNA during recovery appears to reach levels of a greater magnitude than is observed to occur in the presence of galactose (compare Fig. 2 with Fig. 4). The latter is probably due to an increase in epithelial cell differentiation forming new transparent fiber cells [38]. Nath et al. [7] demonstrated that c-myc mRNA elevates in lens cells withdrawing from the cell cycle. Therefore, the higher elevation in c-myc mRNA seen in Figs. 2 and 4 may reflect additive effects, those resulting from: (1) epithelial cell proliferation, and (2) withdrawal of differentiating epithelial cells from the cell cycle. In conclusion, our data strongly suggest that upon exposure of the lens to galactose, c-myc mRNA levels begin to elevate quickly, supporting the concept that the lens epithelial cells respond to the galactose injury by proliferation and differentiation. Our data on

the early increase in MP26 and 7-crystallin mRNAs in lens cells being exposed to galactose support the above implications [36].

Acknowledgements This work was supported by Public Health Service grants EY-05406 to IB and EY-01680 to NJU from the National Eye Institute.

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