Somatic Cell and Molecular Genetics, VoL 19, No. 3, 1993,pp. 285-293

Suppression of Tyrosinase Gene Expression by Bromodeoxyuridine in Syrian Hamster Melanoma Cells Is Not Due to Its Incorporation into Upstream or Coding Sequences of the Tyrosinase Gene Sikha Rauth a,2 and Richard L. D a v i d s o n 2 Specialized Cancer Center and 2Department of Genetics, University of Illinois College of Medicine, 808 South Wood Street, Chicago, Illinois 60612 Received - - 9 April 1993

Abstract--5-Bromodeoxyuridine (BrdU), a thymidine analog, suppresses melanogenesis in Syrian hamster melanoma cells. Tyrosinase, which is the key enzyme for the synthesis of' melanin, is suppressed by exposure to BrdU, and the drop in enzyme activity is correlated with a drop in tyrosinase mRNA level. In order to investigate whether suppression of tyrosinase rnRNA by BrdU is due to BrdU substitution into coding sequences or upstream sequences of the tyrosinase gene, we carried out stable and transient transfection assays' with constructs containing either the human tyrosinase cDNA sequence under the control of a nontyrosinase promoter or a chloramphenicot acetyltransferase (CAT) reporter gene under the control of 5' flanking sequences of the mouse tyrosinase gene. [Unen the plasmid containing the tyrosinase cDNA was stably transfected into mouse fibroblasts, tyrosinase activity in the transfectants was not suppressed by BrdUi Since BrdU would be incorporated into the tyrosinase cDNA integrated in these transfectants, the results suggest that BrdU suppression of tyrosinase gene expression is not due to its incorporation into coding sequences of the tyrosinase gene. When plasmids' with tyrosinase regulatory sequences were transfected into melanoma cells for transient expression assays, CAT gene expression was suppressed by BrdU. Because the CA T plasmids do not contain a mammalian origin of replication and should not replicate under the conditions of transient transfection, BrdU would not be incorporated into the DNA of those plasmids. Therefore, these results suggest that the suppression of tyrosinase gene expression by BrdU also is not due to the incorporation of BrdU into upstream sequences of the tyrosinase gene.

INTRODUCTION

5-Bromodeoxyuridine (BrdU) is a thymidine (dT) analog and is efficiently incorporated into DNA in place of dT. The effects of BrdU have been studied extensively in mammalian systems in relation to the regulation of differentiated functions (1-6). The expression of differentiated functions in a wide variety of cultured cell types (including

chondrocytes, pigment cells, liver cells, etc.) has been found to be suppressed by BrdU. In a few cases, BrdU also has been seen to induce the expression of differentiated functions, for example, prolactin synthesis in pituitary tumor cells (7). In early studies, BrdU-mediated suppression of differentiation in erythroleukemia cells was found to be associated with decreased amounts of globin mRNA (8). In a recent study on the suppres-

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sion of myogenesis by BrdU, it was shown that suppression occurred at the level of the myogenic regulatory gene, MyoD1 (9). In that study, it was shown that BrdU blocks muscle differentiation through the suppression of MyoD1 transcription and not by directly inhibiting the expression of muscle structural genes. However, the specific molecular mechanisms by which BrdU affects differentiation remain to be determined. We and others have shown that BrdU suppresses melanogenesis in melanoma cells (10-14). We also showed that deoxycytidine (dC) reverses BrdU suppression of pigmentation without changing the extent of BrdU incorporation into DNA (12). This observation suggested that the amount of BrdU incorporated into DNA is not the primary determinant per se for suppressing differentiation in melanoma cell. Instead, the results suggested that factors other than BrdU incorporation into DNA were involved in the effect of BrdU on differentiation. Tyrosinase is the key enzyme in the synthesis of melanin (15). In a recent study, we demonstrated that BrdU, at concentrations as low as 0..2-0.4 ~zM, suppressed tyrosinase activity in Syrian hamster melanoma cells by 80-90% and that the level of tyrosinase mRNA was decreased correspondingly (14). These results indicate that BrdU blocks melanogenesis in Syrian hamster melanoma cells through a mechanism that involves suppression of the level of tyrosinase mRNA. Another group also demonstrated that BrdU inhibited tyrosinase activity and tyrosinase mRNA accumulation in mouse melanoma ceils (16). In the present investigation, we carried out experiments to determine whether suppression of tyrosinase gene expression by BrdU is due to BrdU incorporation into either the coding sequence or the upstream regulatory sequences of the tyrosinase gene. In order to test whether BrdU suppression of pigmentation is due to incorporation into the

Rauth and Davidson

tyrosinase coding sequence, we used plasmid pcTYR, containing the full-length human tyrosinase cDNA under the control of the SV40 early region promoter and enhancer. This plasmid has been shown to be stably expressed in transfected mouse fibroblasts (L1), resulting in pigmentation and tyrosinase activity (17). We transfected this plasmid into mouse fibroblasts, isolated stable transfectants, and exposed the transfectants to BrdU under conditions in which BrdU would be incorporated into plasmid DNA, including the coding sequence of the tyrosinase gene. In order to test whether BrdU suppression of pigmentation is due to incorporation into upstream sequences of the tyrosinase gene, we used plasmids containing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of tyrosinase upstream regulatory sequences. With these plasmids, it had been shown that a 270-bp upstream fragment of the tyrosinase gene is sufficient to drive expression of the CAT gene in human and mouse melanoma cells (18). This fragment also has been reported to contain elements for tissue-specific expression in transgenic mice (18). We transfected plasmids with the CAT gene under the control of tyrosinase upstream regulatory sequences into melanoma cells, under conditions of transient transfection, and exposed the transfectants to BrdU under conditions in which BrdU would not be incorporated into ptasmid DNA. Our experiments involving expression of a BrdU-substituted tyrosinase cDNA plasmid suggest that BrdU suppression of pigmentation is not due to BrdU incorporation into the tyrosinase coding sequence. Our experiments involving BrdU suppression of the CAT gene under the control of tyrosinase upstream sequences suggest that BrdU also does not need to be present in the upstream sequences of the tyrosinase gene for suppression of pigmentation to occur.

BromodeoxyuridineSuppression of Melanogenesis

MATERIALS AND METHODS

Cells and Media. The Syrian hamster melanoma cell line W1-1-1 was used in all the experiments involving studies with pigment cells. Line WI-I-1 is a pigmented subclone derived from line Wl-1, which is a stably pigmented subclone of the Syrian hamster melanoma RPMI 3460 (19). Some experiments involved fibroblasts from the murine line LM. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. In the experiments with BrdU, the cells were protected at all times from exposure to wavelengths of light below 550 nm (20) to avoid photodamage to BrdU-containing DNA. Plasmids. Plasmid pcTYR contains the full-length human tyrosinase cDNA under the control of the SV40 early promoter and enhancer (17). Plasmids pSV2CAT and pSVOCAT contain the CAT gene with and without SV40 early promoter and enhancer sequences, respectively (21). Plasmids pTyrCAT (-0.08), pTyrCAT (-0.27), and pTyrCAT (-3.7) contain 80 bp, 270 bp, and 3.7 kb, respectively, of sequences upstream of the transcriptional start site of the mouse tyrosinase gene, cloned into the CAT expression vector pBLCAT6 (18). Plasmid pIRVNeoGal is derived from pIRVNeoAct (22) and contains the [3-galactosidase gene. (Plasmid pIRVNeoGal was constructed by Dr. Cho-Yau Yeung, Department of Genetics, University of Illinois College of Medicine.) Plasmid pZipGptNeo (23) contains both neo and gpt genes and was used in cotransfection experiments for isolation of stable transfectants. Transfection Experiments. For transient transfection assays, WI-I-1 cells were transfected with the pTyrCAT plasmids, using the calcium phosphate precipitation technique (24). Cells were subcultured to achieve 70% confluency in 100-ram tissue cultures dishes and were transfected with 40 Ixg of plasmid

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DNA per dish. In order to investigate the effect of BrdU treatment, cells were pregrown and transfected in DMEM in the presence of 0.4 ~M BrdU, and the cells were maintained in the presence of 0.4 jxM BrdU during the period of plasmid expression following transfection. Plasmids pSVOCAT and pSV2CAT were used in these experiments as negative and positive controls, respectively. Plasmid plRVNeoGaI was cotransfected in all cases, to permit normalization of CAT activity relative to [3-gatactosidase in transfectants. In transient transfection assays, plasmids were allowed to express for 48 h after transfection, and then the cells were collected for enzyme assays. To isolate cells stably transfected with plasmid pcTYR, LM cells were cotransfected with plasmids pcTYR and pZipGptNeo. Three days after transfection, the cells were subcultured in the presence of 1 mg/ml G418 (Sigma Chemical Co.), to select for cells expressing the neo gene. The selective medium with G418 was replaced every three days, and coIonies that appeared after 10-14 days were pooled and subcultured tbr further analysis. CATAssay. For analysis of CAT activity, transfected cells were collected by centrifugation and then homogenized and sonicated in homogenizing buffer as previously described (25). After centrifugation, the clear supernatants were assayed for protein concentration by the Bradford method (26), using the Bio-Rad protein assay kit. The samples were heated at 70°C for 10 rain to inactivate inhibitors of CAT activity, as previously reported (25). The assay conditions and chromatographic methods used were essentially the same as previously described (25). The chromatograms were scanned using a Betascope 603 blot analysis to quantitate acetylated forms. Measurements of CAT activities were normalized for variations in transfection efficiency by measuring ~3-galactosidase activity (see below) and

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using for the CAT assay an amount of extract that contained a standard level of [3-galactosidase activity. In general, within and between experiments, the transfection efficiency as indicated by ~-galactosidase activity varied only minimally. CAT activity is expressed as percent conversion to acetylated derivatives per hour. ~-GalactosidaseAssay. Extracts of transfected cells that were used to assay CAT activity also were assayed for 13-galactosidase activity, as an internal reference in transfection studies. [3-Galactosidase activity is sensitive to heat, and so extracts were tested for [3-galactosidase activity prior to heating for CAT assays. Cellular extracts containing 200 txg of protein were assayed for [3-galactosidase activity in each case. TyrosinaseAssay. Tyrosinase activity was measured by a sensitive radiometric assay, based on tyrosinase hydroxylase activity, and was determined as previously described (27). The assay procedure involves incubating cell extracts with [3H]tyrosine (and DOPA as required cofactor) in a sodium phosphate buffer and measuring the release of 3H20 produced by the tyrosinase hydroxylase activity of the enzyme. All assays were performed in duplicate. Tyrosinase activity was calculated as follows: (3H20 release by test cell lysate) - (3H20 release by control) and expressed as cpm 3HzO/min/mg protein. Measurement of BrdU Substitution in Genomic DNA. BrdU incorporation into the genomic DNA of Syrian hamster melanoma cells and mouse fibroblasts cells was measured by a procedure developed and standardized by Dr. Elliot Kaufman, Department of Genetics, University of Illinois College of Medicine (unpublished results). Tissue culture dishes, 150 mM, were inoculated with 2 x 106 cells in 50 ml of DMEM medium plus 0.4 FxM BrdU. The cultures were refed with the same medium after three days and harvested two days later. Genomic DNA was prepared from cells and then enzymaticaIly digested to its component 5'-deoxymononu-

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cleosides in the following manner. To 1 ml of DNA solution, 0.1 ml of 0.1 M MgC12 and 50 ~I of DNase I (5 mg/ml stock solution) were added, and the DNA was digested for 1 h at 37°C. An additional 25 ~1 of DNase I stock solution was added, and a further incubation at 37°C was carried out for 30 min. Deoxyoligonucleotides were produced by the addition of 0.1 ml of 1 M glycine, pH 9.2, and 10 ixl of snake venom phosphodiesterase (10 mg/ml stock solution) to the DNA solution, and the solution was incubated for 1 h at 37°C. An additional 5 txl of phosphodiesterase stock solution was added, and the reaction continued for another 30 rain at 37°C for complete digestion of DNA. Finally the 5'-deoxynucleosides were produced by adding calf intestinal alkaline phosphatase (0.25 units) and incubating for 30 rain at 37°C. The 5'-deox3*nucleosides were separated by high-pressure liquid chromatography using a reverse-phase column, and they were quantitated by comparison to known standards. RESULTS

Effect of BrdU on Tyrosinase cDNA Expression. It has been demonstrated that stable transfection of the full-length human tyrosinase cDNA under the control of SV40 sequences (in plasmid pcTYR) can lead to tyrosinase activity and pigmentation in mouse fibroblasts that do not normally synthesize melanin (17). In order to determine the effect of BrdU incorporation into the tyrosinase coding sequence, we isolated mouse fibroblast LM cells stably transfected with plasmid pcTYR (by cotransfection with plasmid pZipGptNeo and selection for G418 resistance). As shown in Table 1, the tyrosinase activity of a pool of stably transfected cells was approximately one third as high as the tyrosinase activity of the highly pigmented Syrian hamster melanoma cell line W1-1-1, whereas untransfected LM cells showed negligible activity. The effect of BrdU on the expression of

BromodeoxyurldineSuppression of Melanogenesis

Table 1. Effect of BrdU on Tyrosinase Activity of Mouse Fibroblasts Stably Transfected with Human Tyrosinase cDNA

Cell line

Plasmid transfected

Melanoma Wl-l-1 Fibroblasts LM Fibroblasts LM

--pcTYR

Tyrosinase activit~~ -BrdU

+BrdU

122 2 43

8 2 36

aMelanoma cells, fibroblasts, and stably transfected fibroblasts were grown in the presence or absence of 0.4 txM BrdU for five days. The cells were harvested and extracts prepared for tyrosinase assays. Tyrosinase activity is expressed as cpm 3H20/min/mg protein of cellular extract.

the tyrosinase cDNA in the stable transfectants was determined by growing the cells in the presence or absence of 0.4 ~M BrdU for five days. [We had previously shown that such tow concentrations of BrdU have minimal effects on cell growth (12, 14).] With Syrian hamster melanoma cells, pigmentation decreased markedly as a result of BrdU treatment, and the BrdU-treated melanoma cells showed approximately a 95% drop in tyrosinase activity relative to untreated melanoma cells. In contrast, with the pcTYR transfected LM ceils, BrdU treatment resulted in only a minimal decrease (approximately 15%) in tyrosinase activity. In fact, even though the untreated melanoma cells had approximately three times higher tyrosinase activity than untreated pcTYR transfected fibroblasts, the tyrosinase activity in BrdU-treated melanoma cells was almost five times lower than in the BrdU-treated, transfected fibroblasts. To assess the difference in BrdU response in the two cell lines, we measured BrdU incorporation into genomic DNA of the transfected mouse fibroblasts and Syrian hamster melanoma cells. The results are expressed as BrdU substitution, the percent replacement of dT by BrdU in nuclear DNA. In the BrdU-treated Syrian hamster melanoma cells, BrdU substitution was 13%. In the BrdU-treated, transfected fibroblasts it

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was 11%. Presumably, the extent of BrdU replacement for dT in bulk genomic DNA in the two cell lines reflects BrdU incorporation into the endogenous tyrosinase gene and the transfected tyrosinase cDNA. Since BrdU substitution was almost the same in the transfected mouse cells, in which BrdU had only a minimal effect on tyrosinase activity, and in the melanoma cells, in which BrdU almost totally abolished tyrosinase activity, the results suggest that incorporation of BrdU into the coding region of the tyrosinase gene per se is not sufficient to suppress tyrosinase gene expression. Effect of BrdU on CA r Gene Expression. It has been shown that a 270-bp upstream fragment of the tyrosinase gene is sufficient to drive gene expression in human and mouse melanoma cells (18). In our studies, we transfected the pTyrCAT contructs into Syrian hamster melanoma cells W1-1-1 and also into mouse fibroblasts LM, which do not express detectable levels of tyrosinase mRNA. These experiments were carried out using transient expression assays. As shown in Table 2, transfection of both plasmids pTyrCAT (-0.27) and pTyrCAT (-3.7) into melanoma cells led to a high level of CAT activity. (The plasmid with 3.7 kb of upstream tyrosinase gene sequence generated a Table 2. Expression of pTyrCAT Plasmids in Syrian Hamster Melanoma Cells and Mouse Fibroblasts CAT activity ~

Ptasmid transfected

Melanoma

Fibrobtasts

pSVOCAT pSV2CAT pTyrCAT(-0.08) pTyrCAT(-0.27) pTyrCAT(-3.70)

1 91 9 46 70

1 32 3 5 2

aMelanoma cells and fibroblasts were transfected for transient expression assays, using 40 lxg of CAT plasmid plus 20 ~g of plasmid pIRVNeoGal per dish. After 48 h for plasmid expression, the cells were harvested and extracts prepared for CAT and [3-galactosidase assays. Extracts were normalized for 13-gatactosidase activi~. CAT activity is expressed as percent conversion to acetylated forms per hour.

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CAT (-0.27) and pTyrCAT (-3.7) in melanoma cells dropped significantly in the presence of BrdU. CAT activity of the pSV2CAT plasmid in the same experiment remained the same in the presence and absence of BrdU (Table 3). The melanoma cells grown in the absence of BrdU appeared highly pigmented, whereas, in the presence of 0.4 ~xM BrdU, the cells were only slightly pigmented. In the presence of BrdU, the tyrosinase activity of the cells dropped significantly, both in the transfected and untransfected cells (approximately 95% drop in both cases). Thus, transfection itself had no effect on the extent of BrdU suppression of the endogenous tyrosinase gene. In these experiments, BrdU suppressed the expression of plasmid pTyrCAT (-3.7) approximately as much as it suppressed the expression of the endogenous tyrosinase gene, and the suppression of plasmid pTyrCAT (-0.27) appeared less extensive. However, in other experiments, BrdU suppressed the expression of plasmid pTyrCAT (-0.27) by approximately 95%, like the endogenous tyrosinase gene. The pTyrCAT plasmids do not contain a mammalian origin of replication and should not replicate under the condition of transient expression assays (29-31). Therefore, BrdU Table 3. Effectof BrdU on CAT and TyrosinaseGene should not be incorporated into plasmid Expression in MelanomaCells DNA under such conditions. Although we CAT Tyrosinase have not demonstrated that the transfected activity" activity" plasmids did not undergo replication in our Plasmid experiments, it is reasonable to assume that transfected -BrdU +BrdU -BrdU +BrdU plasmid replication did not occur and that we pSV2CAT 94 94 197 11 were measuring CAT gene expression of pTyrCAT (-0.27) 43 12 190 8 BrdU unsubstituted plasmids isolated from pTyrCAT BrdU-treated cells. These results suggest (-3.7) 20 2 172 6 that BrdU suppression of CAT gene expresNone 2 2 185 7 sion under the control of upstream tyrosinase aSyrian hamster melanoma cells were pretreated with gene sequences does not require the incorpo0.4 b~MBrdU for fivedaysand maintainedin BrdU during transfectionand expressionof the plasmidsfor two ration of BrdU into the tyrosinase gene days. The cells also were cotransfected with plasmid pIRVNeoGal. Cellular extracts were prepared and as- regulatory sequences. sayed for CAT, [3-galactosidase,and tyrosinase activiEffect of BrdU Treatment for Different ties. CAT and tyrosinaseactivitiesare expressed as in- Time Periods. In order to determine how dicated in Tables 1 and 2. long cells needed to be exposed to BrdU for

higher level of CAT gene expression in this experiment than the plasmid with 270 bases of upstream sequences. However, this was not reproducibly observed in other experiments.) No significant CAT activity was found with any of the pTyrCAT plasmids transfected into mouse fibroblasts. Plasmid pSV2CAT expressed a high level of CAT activity in both transfected melanoma and fibroblasts cells. These results indicate that a 270-bp fragment 5' of the transcriptional start site of the tyrosinase gene is sufficient to drive gene expression in Syrian hamster melanoma cells, as had been shown previously with human and mouse melanoma cells (18). In order to test whether BrdU-mediated suppression of tyrosinase gene expression is due to incorporation of BrdU into upstream sequences of the tyrosinase gene, the pTyrCAT constructs were transfected into BrdUtreated melanoma cells under conditions of transient expression assays. In the initial experiments, the ceils were treated with 0.4 ~M BrdU for five days prior to transfection and maintained in BrdU during exposure to DNA and for a two-day period (for CAT gene expression) thereafter. As shown in Table 3, CAT activity with plasmids pTyr-

Bromodeoxyuridine Suppression of Melanogenesis

suppression of gene expression to occur, the Syrian hamster melanoma cells were pretreated with 0.4 IxM BrdU for one to five days prior to transfection with the plasmid pTyrCAT (-0.27). In all cases, except one (see Table 4), the cells were maintained in the presence of BrdU during exposure to plasmid DNA and for 48 h after transfection for CAT gene expression. In cells treated with BrdU for one day prior to transfection, CAT activity was approximately 50% as high as in untreated cells (Table 4). CAT activity in cells pretreated with BrdU for two or three days was lower, but only after five days of BrdU pretreatment was suppression of CAT activity almost complete (approximately 95%). The removal of BrdU from the medium during the two-day expression period after transfection of the CAT plasmid resulted in the complete abolition of BrdU suppression of CAT gene expression, even though the cells had been pretreated with BrdU for five days and were transfected in the presence of BrdU. These results indicate that BrdU treatment of cells for several days prior to transfection of plasmid pTyrCAT (-0.27) is necessary for BrdU suppression of CAT gene expression, and also that BrdU Table 4, Effect of Timing of BrdU Treatment on CAT

Gene Expression BrdU pretreatment time (days)a

BrdU during expression timeb

CAT activity~

-

-

80

0 1 2 3 5 5

+ + + + + -

83 38 17 18 4 80

~Syrian hamster melanoma cells were pretreated with 0.4 ~M BrdU for different time periods prior to transfection with plasmids pTyrCAT (-0.27) and plRVNeoGal. bOA ixM BrdU was present in the medium during the transfection period (overnight exposure to plasmid DNA), The presence or absence of BrdU during the subsequent two-day plasmid expression period is indicated. CCAT activity is expressed as indicated in Table 2,

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needs to be maintained in the medium during the expression period following transfection for suppression to be observed.

D I S C U S S I O N

We have shown previously that BrdU suppresses tyrosinase activity in Syrian hamster melanoma cells and that suppression is associated with a marked decrease in the level of tyrosinase mRNA (14). One possible explanation for the effect of BrdU is that suppression is due to incorporation of BrdU into the coding sequence of the tyrosinase gene. To test this, we analyzed the effect of BrdU on the expression of the tyrosinase cDNA in plasmid pcTYR (containing the full-length human tyrosinase cDNA under the control of SV40 sequences) when the plasmid is stably transfected into mouse fibroblasts. Our results showed that plasmid expression (as measured by tyrosinase activity) was essentially the same in the presence or absence of BrdU. Since the ptasmid was stably transfected into the mouse cells, the results presumably reflect the expression of BrdU-substituted tyrosinase cDNA sequences in plasmids integrated into the mouse genome. We measured BrdU substitution in the genome of the pcTYR-transfected mouse fibroblasts and also Syrian hamster melanoma cells and found the extent of BrdU substitution to be essentially the same in both cases. Since BrdU did not cause a significant drop in tyrosinase activity in pcTYR-transfected mouse fibroblasts, the results suggest that incorporation of BrdU into the coding sequence of the tyrosinase gene is not incompatible with tyrosinase gene expression. Assuming that fibroblasts contain the same BrdU-responsive elements as do melanoma cells, we conclude that BrdUmediated suppression of the endogenous tyrosinase gene in Syrian hamster melanoma cells is not due to its incorporation into the coding sequences of the tyrosinase gene. We also determined the effect of BrdU

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on the expression of the CAT gene in dependence for the effects of BrdU to be pTyrCAT plasmids transfected into mela- observed. Syrian hamster melanoma celis noma ceils for transient expression assays. apparently need to be exposed to BrdU for These plasmids contain the CAT gene under several days prior to transfection of the the control of upstream sequences of the pTyrCAT plasmids in order to provide the tyrosinase gene. In the presence of 0.4 p~M cellular environment for complete BrdU BrdU, CAT activity in the transfected cells suppression of CAT gene expression. In dropped significantly (as did the activity of contrast to the long period of BrdU pretreatthe endogenous tyrosinase gene). In con- ment required for complete suppression to trast, 0.4 txM BrdU had no effect on CAT occur, the reversal of suppression seems to gene expression with plasmid pSV2CAT occur very rapidly. When cells were pretransfected into melanoma cells. These re- treated with BrdU for five days and BrdU sults suggest that BrdU-mediated suppres- was removed after the cells were transfected, sion of CAT gene constructs transfected into a period of two days for plasmid expression melanoma cells involves regulation that is in the absence of BrdU was sufficient to tyrosinase gene specific and is mediated via permit full expression of the CAT gene in the the upstream tyrosinase gene sequences in plasmid. The reasons for the requirement for the plasmids. (The suppression of CAT gene BrdU to be present during the period of expression by BrdU in these experiments plasmid expression (even though BrdU apparalso agrees with the conclusion :from the ently is not incorporated into plasmid DNA experiments discussed above--namely, that in these experiments) and for the rapid the tyrosinase gene coding sequence per se is restoration of plasmid expression upon the not involved in BrdU suppression of differen- removal of BrdU remain to be elucidated. The results of our studies on the effect tiation in melanoma cells.) The results with the pTyrCAT plasmids of BrdU on tyrosinase gene regulatory raise the question whether BrdU suppression sequences are in agreement with those of of CAT gene expression is due to the in- comparable studies on the myosin light chain corporation of BrdU into the upstream 2 promoter (32). When plasmids with the tyrosinase gene sequences in these plasmids. CAT gene under the control of the myosin However, in these experiments, the plasmids promoter were transfected (for transient were transfected under conditions of tran- expression assays) into muscle cells, it was sient expression and presumably there was found that BrdU did not need to be no opportunity for chromosomal integration incorporated into the myosin promoter in the and replication of the plasmids to occur in plasmid for suppression of CAT gene expresthe presence of BrdU. Under such condi- sion to occur. The precise mechanisms by which BrdU tions, the CAT activity measured in these suppresses gene expression remain to be experiments should reflect the expression of elucidated. It has been suggested that the plasmids that did not contain BrdU in their DNA. Thus, the BrdU-mediated suppression effect of BrdU on muscle cells is due to of CAT gene expression observed in these suppression of expression of the myogenic experiments apparently is not dependent activator gene MyoD1 rather than suppresupon the incorporation of BrdU into the sion of the muscle structural genes directly upstream tyrosinase gene sequences in the (9). Our results on the effects of BrdU on plasmids, although BrdU suppression is plasmid gene expression driven by tyrosinase dependent upon the presence of these gene regulatory sequences are compatible with such a mechanism. Although a regulasequences. Our results also indicate a marked time tory gene comparable to MyoD1 has not been

BromodeoxyuridineSuppression of Melanogenesis

demonstrated for the tyrosinase gene, it is conceivable that such a regulatory gene exists and that the effect of BrdU on melanogenesis involves suppression of such a tyrosinase regulatory locus. Thus, the studies on BrdU suppression of differentiation in melanoma cells could lead to the identification of such a t3'rosinase regulatory gene. ACKNOWLEDGEMENTS We thank Dr. Brigitte Bouchard, Dr. Alan Houghton, and Dr. Gunther Schutz for kindly providing us with the pcTYR and pTyrCAT constructs used in our studies. We also thank Dr. Elliot Kaufman for performing BrdU incorporation studiesl Dr. ChoYau Yeung for providing plasmid pIRVNeoGal, and Dr. Mark Kresnak for providing LM cells stably transfected with the tyrosinase cDNA plasmid. This work was supported by Public Health Service grant CA31777 from the National Institutes of Health. LITERATURE CITED 1. Rutter, W.J., Pictet, R.L., and Morris, P.W. (1972). Annu. Rev. Biochem. 42:601-646. 2. Stockdale, F., Okazaki, K., Nameroff, M., and Holtzer, H. (1964). Science 146:533-535. 3. Bischoff, R., and Holtzer, H. (1970). J. Cell Biol. 44:134-150. 4. Weintraub, H., Campbell, G.L., and Holtzer, H. (1972). J. Mol. Biol. 70:337-350. 5. Abbot, J., and Holtzer, H. (t968). Proc. Natl. Acad. Sci. U.S.A. 59:1144-1151. 6. Saxe, S.A., Lukens, L.M., and Pawlowski, P.J. (1985). J. Biol. Chem. 260:3812-3819. 7. Biswas, D., Lyons, J., and Tashjian, A. (1977). Cell 11:431-439. 8. Preisler, H., Housman, D., Scher, W., and Freind, C. (1972). Proc. Natl. Acad. Sci. U.S,A. 70:29562959.

293 9. Tapscott, J.S., Lasser, A.B., Davis, R.I., and Weintraub, H. (1989). Science 245:532-536. 10. Silagi, S., and Bruce, S.A. (1970). Proc. Natl. Acad. Sci. U..SM. 66:72-78. 11. Wrathall, J., Newcomb, E., Balint, R., Zeitz, L., and Silagi, S. (1975). J. Phys. 86:581-592. 12. Davidson, RA~, and Kaufman, E.R. (1977). Cell 12:923-929. 13. Reff, M.E., and Davidson, R.L. (1979). J. Biol. Chem. 254:6869-6872. 14. Rauth, S., Hoganson, G.E., and Davidson, R.L. (1990). Somat. Cell Mol. Genet. 16:583-592. 15. Pomerantz, S.H. (1964). Biochem. Biophys. Res. Commun. 16:t88-194. 16. Kidson, S.H., and DeHaan, J.B. (1990). Exp. Celt Res. 188:36-41. 17. Bouchard, B., Fuller, B., Vijayasaradhi, S., and Houghton, A. (1989). J. Exp. Med. 169:2029-2042. 18. Kluppel, M., Beermann, F., Ruppert, S., Schmid, E., Hummler, E., and Schutz, G. (1991). Proc. Natl. Acad. Sci. U.S.A. 88:3777-3781. 19. Moore, G.E. (1964). Exp. Celt Res. 36:422-423. 20. Kaufman, E.R., and Davidson, R.L. (1977). Exp. Cell Res. 107:15-24. 21. Gorman, C.M., Moffat, L.F., and Howard, B.H. (1982). Mol. Cell Biol. 2:1044-1051. 22. Morganstein, J.P., and Land, H. (1990). Nucleic Acids Res. 18:3587-3596. 23. Ashman, C.R., Jagadeeswaran, P., and Davidson, R.L. (1986). Proc. Natl. Acad. Sci. U.S.A. 83:33563360. 24. Wigler, M., Sweet, R., Sui, G.K., Wald, B., Pellicer, A., Lacy, R., Maniatis, T., Silverstein, S., and Axel, R. (1979). Cell 16:777-785. 25. Rauth, S., Yang, K.G., Seibold, A.M., Ingolia, D.E., Ross, S.R., and Young, G.Y. (1990). Somat. CellMol. Genet. 16:129-141. 26. Bradford, M.M. (1976). Anat. Biochem. 72:248254. 27. Pomerantz, S.M. (1969). Science 164:838-839. 28. Biamoniti, G., Della Valle, G., Talarico, D, Cobianchi, F., Riva, S., and Falaschi, A. (1985). NucIeic Acids Res. 16:5545-556L 29. Umek, R.M., Linskens, M.H.K., Kowalski, D., and Huberman, J.A. (1989). Biochim. Biophys. Acta 1007:1-14. 30. Dean, F.B., Borowiec, J.A., Ishimi, Y., Del, S,, Tegtmeyer, P., and Hurwitz, J. (1.987). Proc. Natl. Acad. Sci. U.S.A. 84:8267-8271. 31. Gutierrez, C., Gino, Z., and Burhaus, W. (1988). Science 240:1202-1203. 32. Arnold, H.H., Tannich, E., and Paterson, B.M. (1988). Nucleic Acids Res. 16:2411-2428.