The effect of short term incubation in vitro on the RNA content of regenerating mouse epidermis.код для вставкиСкачать
THE ANATOMICAL RECORD 196~349-353 (1980) The Effect of Short Term Incubation In Vitro on the RNA Content of Regenerating Mouse Epidermis MARGARET ANNE TORMAY AND THOMAS S. ARGYFtIS Department of Pathology, Upstate Medical Center, SUNY, Syracuse, New York ABSTRACT The effect of short term incubation in vitro on RNA content in regenerating and normal epidermis has been investigated. Regenerating mouse epidermis was incubated for three hours, either attached to its underlying dermis or by itself, i n either buffered sucrose or Roswell Park Memorial Institute culture medium at 26°C or at 37°C. There is a significant loss of RNA when regenerating epidermis is incubated without being attached to its underlying dermis, a t either 26°C or 37"C, although there was little loss of DNA, good incorporation of ("HI orotic acid into RNA, as well as good preservation of epidermal histological details. In contrast, when regenerating epidermis was incubated attached to its dermis, little loss of RNA occurred. Similarly, incubating normal epidermis attached to its dermis results i n no loss of RNA. These conditions also result in no significant loss of DNA, good incorporation of ("HI orotic acid into RNA, and preservation of epidermal histological details. The study of epidermal RNA synthesis i n vitro has increased considerably (Flamm et al., '66; Delapp and Karasek, '76). Usually much attention is paid to make certain that there is a linear incorporation of the appropriate precursors into RNA (Flamm et al., '66; Delapp and Karasek, '76) under the conditions of study in vitro. To the best of our knowledge, no attention has been paid to whether or not the in vitro incubation of the epidermis results in a loss of RNA and if so, if i t is related to a change in the histological appearance of the epidermis. Because we have been interested in the role of the RNA, which accumulates during epidermal regeneration in the regulation of the epidermal hyperplastic growth (Argyris, '76; '77; '78), and in skin carcinogenesis (DeYoung et al., '77; Mueller and Argyris, '77), we have been seeking conditions for the short term study of RNA metabolism of the hyperplastic epidermis in vitro. This report will demonstrate that the incubation of regenerating epidermis for as little as three hours in vitro can result in the loss of considerable RNA, even though there is a vigorous incorporation of precursor into RNA. Moreover, the loss of the RNA is not reflected in any change in the histological appearance of the regenerating epidermis. We will also show that under some incubation conditions, we can prevent the loss of much of the RNA if the 000-3276X/80/1963-0349$01.400 1980 ALAN R. LISS, INC. regenerating mouse epidermis is left attached to its dermis. MATERIALS AND METHODS Mice and abrasion technique CD-1 female mice, 40 days of age, were purchased from Charles River Farms, Wilmington, Mass. The conditions for maintenance of the mice has been already described (Argyris, '76; '77). Epidermal abrasion was performed by using a felt wheel mounted on a Dremel mototool as previously described (Argyris, '76; '77). Media Sucrose. 0.25 M was made in 0.02 M Trizma (pH 7.5, 20°C), 0.25 M KC1, 0.005 M MgC12, and 0.001 M dithiothreitol (STKM). Roswell Park Memorial Institute tissue culture medium 1640 (RPMI) was obtained from Dr. Bertie F. Argyris, Department of Microbiology in this institution. The contents of the medium are described in Argyris, B. ('77), except we did not add serum to the medium. Preparation of epidermis for in uitro study Regenerating epidermis was studied in vitro in two different ways. It was placed in vitro, attached to its underlying dermis (epidermis Received June 21, 1979, accepted September 4, 1979 350 MARGARET ANNE TORMAY AND THOMAS S. ARGYRIS plus dermis), or separated from its underlying dermis (epidermis alone). For the incubation in vitro of regenerating epidermis with its dermis, 8 abraded mice were sacrificed by cervical dislocation. The backs clipped, the depilatory Surgex applied for two minutes and rinsed off with running water (Argyris, '77). The abraded area was cut out and placed in saline at 4°C. The subcutaneous tissues were scraped away (Argyris, '77) and the regenerating epidermis, along with its underlying dermis, was placed i n either 0.25M STKM or RPMI, in plastic tissue culture dishes, 60 x 15 mm (Falcon Plastics, Los Angeles, CA.). Usually, two wounds were divided in half, each piece was about 1.5 cm2, and the four pieces were placed in a plastic culture dish. Each piece contained about 12 mg of epidermis. For each determination, the epidermis from the 4 halves was pooled. The incubation period was for 3 hours, either at room temperature (26"C), or at 37"C, in a CO, incubator. To the medium, 25 pCi/ml of (5-"H)orotic acid (Schwarz/Mann, Orangeburg, N.Y.) Sp.Act. 10 Ci/mM, at a concentration of 0.5 mCi/ml, was added. Total volume of the incubation mixture was 4 ml. Following the 3 hours incubation period, the wounds were removed and washed 3 times with ice cold saline. The regenerating epidermis was then scraped off with a scalpel, dried between 2 layers of No. l W h a t m a n filter paper, and weighed (Argyris, '77). For incubation of regenerating epidermis alone in vitro, wounds were cut out from mice, and the subcutaneous tissues removed by scraping as described above. The wounds were then placed dermis side down on top of a n ice filled Petri dish, and the regenerating epidermis scraped off as previously described (Argyris, '77). The epidermis from 2 wounds was quickly washed in 3 changes of ice cold saline and placed in a Falcon dish with the appropriate medium. Upon completion of the incubation period, the regenerating epidermis was removed, rinsed with 3 changes of ice cold saline, and dried as described above. Normal epidermis attached to its underlying dermis was also incubated in vitro for 3 hours a t 26"C, either in 0.25 M STKM or RPMI, with 25 pCi/ml of (5-"H)orotic acid. Total volume of the incubation mixture was also 4 ml. Four pieces of normal epidermis plus dermis, cut to approximate the size of the pieces of regenerating epidermis plus dermis, were placed in each dish. Each piece had about 8 mg of epidermis. The epidermis from the 4 pieces was pooled for each determination. Preparation of homogenates Epidermal homogenates 4% w/v i n 0.25 M STKM were prepared before or after incubation as already described (Mueller and Argyris, '75). Acid soluble material (ASM), RNA, and DNA were obtained by extracting 1 m l of t h e homogenates using a Schmidt-Thannhauser procedure (Mueller and Argyris, '75). RNA was measured using lAZBO units = 32 pgRNA/ml (Argyris, '78). DNA was determined by measuring the A,,,, using purified calf thymus DNA (Sigma Chemical Co., St.Louis, MO.) as a standard. The values were similar to those obtained using the diphenylamine reaction (Argyris, '78). The amount of radioactivity in the epidermal homogenates and in the ASM and RNA fractions from the Schmidt-Thannhauser extraction was determined by placing 0.2 ml of the homogenate or of the ASM and RNA fractions i n a scintillation vial, to which was added 1ml of NCS solubilizer (AmershamiSearle, Arlington Heights, IL.) and 1 0 m l of the premixed scintillation cocktail, Omnifluor (New England Nuclear, Boston, Mass.) dissolved in reagent grade toluene. Sample counting was done in a Packard Tri-Carb scintillation spectrometer, model 3320. Counting efficiency was 3Wo. Background ranged from 11-22 cpm. Biopsy specimens of epidermis and dermis, or epidermis alone, from normal and abraded skin were taken before and after the 3-hour incubation period, fixed in b d e r e d formalin, and processed for routine histological study (Argyris, '76). Statistical analysis was done using Student t test. P values of 0.05 or less were considered significant. RESULTS We first have determined whether or not the 3-hour incubation period results in a net loss of epidermal RNA, DNA, and acid soluble material (ASM). Table I indicates the values for RNA, DNA, and ASM of regenerating epidermis in intact normal mice. Epidermis incubated alone in vitro at 26°C shows a significant decrease in RNA. The greatest loss of RNA is in epidermis incubated alone a t 37°C in RPMI (Table 2). The least loss of epidermal RNA in vitro is seen (Table 2) in epidermis plus dermis incubated at 26°C i n STKM, which shows a n RNA value which is 94%of that in intact mice. Incubation of epidermis plus dermis i n RPMI also results in only a small, but not significant loss of RNA (Table 2). The concentration of the ASM increases moderately over that which ap- 35 1 EPIDERMAL RNA IN VITRO TABLE 1. RNA, DNA, and acid soluble material (ASMI of normal and regenerating female mouse epidermis ~~ RNA ASM (A,,, unitsig epidermis) (6)" 1.65 i 0.035 2.98 t 0.14 (6) 4.54 2 0.076 34.1 2 4.22 (6) 28.8 & 2.12 (8) (7) (9) (mgi&$ZFmis) Abraded t 7 days Normal ~~ DNA (mgigmis) 4.66 & 0.22' *Average t standard error of the mean. "umber of determinations in parentheses. pears in vivo, depending on the conditions of incubation (Table 2). The DNA content is not significantly affected i n any of the incubation conditions (Table 2). Histological preservation is quite good under all conditions of incubation (not shown). The incorporation of (") orotic acid into RNA and ASM is indicated in Table 3. It is clear that the best incorporation of ("HIorotic acid into RNA is in the epidermis incubated i n RPMI either alone or attached to its dermis. Within this group in turn, the highest specific activity of RNA is for epidermis incubated alone at 37°C-the condition, interestingly enough, which shows the greatest net loss of RNA. The incorporation of ('<HIorotic acid into the ASM is also greatest in epidermis cultured alone in RPMI (Table 3). For comparison, we have also studied the incorporation of (:iH) orotic acid into RNA and ASM of normal mouse epidermis in vitro. We have limited our study to normal epidermis attached to its dermis, incubated a t 26"C, either in STKM or RPMI, because these are the conditions which give the best results for regenerating epidermis. In Table 4 it is seen that the values for RNA, DNA, and ASM after the 3-hour incubation period at 2 6 T , in either sucrose TKM or RPMI, are quite close to the values seen in intact mice (Table 1).Table 4 also indicates that the best incorporation of (:jH)orotic acid into RNA is in the RPMI medium. Under both conditions of study, there is very good preservation of histological detail (not shown). DISCUSSION The best preservation of RNA is obtained when the regenerating epidermis is incubated attached to its dermis at 26°C in buffered sucrose. Almost as good RNA preservation is obtained in RPMI medium under similar conditions. Increasing the temperature to 37°C results in a considerable loss of RNA in the regenerating epidermis incubated i n either STKM or RPMI. Incubating the regenerating epidermis without its underlying dermis also results in a significant decrease in RNA, which becomes even more severe when the incubation is done at 37°C. The DNA of the regenerating epidermis is minimally affected by either increasing the temperature or incubating the epidermis without its dermis. The amount of acid soluble material as measured by the number of A,,, units/g epidermis appears to rise moderately in all conditions of incubation. The incorporation of ('jH) orotic acid into RNA in regenerating epidermis incubated in vitro under conditions of maximal preservation of RNA, that is when the regenerating epidermis attached to its dermis is incubated at 26"C, in either STKM or RPMI, is quite good. However, the highest specific activity of RNA in the regenerating epidermis occurs when the epidermis is incubated alone at 37°C in RPMI medium. Calculations indicate that this increased specific activity of RNA cannot be accounted for solely by the increase i n the specific activity of the acid soluble pool. Interestingly, incubation of epidermis alone in RPMI results in the greatest loss of RNA. This raises the possibility that the higher specific activity of the RNA in the epidermis incubated in RPMI medium at 37°C may be a n artefact, because the RNA which was lost was the RNA which had already been synthesized prior to labelling the epidermis with ("H) orotic acid, and therefore, was not labelled. This would make comparisons of RNA specific activity with the specific activity of RNA under conditions in which little, if any, RNA is lost difficult, because the lower specific activity in the regenerating epidermis could be due to the fact that the cells of the regenerating epidermis contain considerable amounts of unlabelled RNA, which would lower the value for the specific activity of the RNA. We, therefore, calculated the incorporation of H")( orotic acid into RNA per mg DNA. The cpm in epidermal RNA, when calculated per mg DNA, show a considerable reduction in RNAC DNA ASMd RNA DNA ASM Condition cpm mgRNA CPm A,=its Cpm mgRNA CPm A.,,,,=its "Average r standard error of the mean. "Number of determinations In parentheses Epidermis and dermis Epidermis alone epidermis of 2.50 ? 0.150 (8) 2.15 ? 0.130 (8) 56.2 t 5.60 (8) 2.75 t 0.150 (9) 2.15 -c 0.110 (9) 61.0 ? 4.40 (9) 0.25 M Sucrose - TKM 37°C 3.27 ? 0.470a (13P 2.98 ? 0.120 (13) 49.3 -c 3.60 (13) 4.38 t 0.140 (8) 2.75 i- 0.160 (11) 65.2 t 4.30 (11) 26°C 1.25 t 0.140 (5) 2.43 f 0.0070 (5) 61.7 -c 10.7 (5) 3.67 t 0.170 (10) 2.87 f 0.230 (10) 49.9 c 2.70 (10) 26°C 37°C 8,400 t1,300 (9) 11,700 i- 800 (6) (8) (8P 31,900t 5,000 22,900? 5,700 (8) 7,600 t 600 (9) 5,600 f 900 (9) (5) 43,300 -c 12,000 (5) 32,400 ? 7,800 (10) 15,300 -c 1,600 (10) 57,700 t 12,200 9,800 f 800 (8) 26°C 37°C 0.25 M. Sucrose - TKM 18,500-c4,7OOd 26°C RPMI (6) 29,500 ? 9,100 53,400(6) 2 6,300 125,600 t 24,500 37°C 1.11? 0.080 (8) 2.45 ? 0.150 (8) 47.4 f 3.9 (8) 2.84 t 0.220 (8) 2.96 -c 0.230 (8) 65.8 ? 4.60 (8) RPMI TABLE 3 . Incorporation of (5:'H) orotic acid into RNA and ASM of regenerating female mouse epidermis 7 ahys after abrasion, incubated in vitro for 3 hours ^Average 2 standard error of the mean. "Numberof determinations in parentheses. 'RNA and DNA, mgig epidermis. dASM, A,,,, uniWg epidermis. Epidermis and dermis Epidermis alone Condition of epidermis TABLE 2. The RNA, DNA, and ASM content of regenerating female mouse epidermis 7 days after abrasion incubated in vitro for 3 hours 4 E 0 2 U 4 5 5 z 0 3e 5 e E i EPIDERMAL RNA IN VITRO 353 TABLE 4. Incorporation of (&'HI orotic acid into epidermal RNA and ASM, and the epidermal RNA, DNA, and ASM content of normal female mouse epidermis plus dermis incubated in uitrv far 3 hours Measurement 0.25 M Sucrose - STKM RPMI RNA (mgig epid.) DNA (mgig epid.) ASM (A,,,,units/g) 1.44 2 0.080 (8) 4.10 2 0.40 (8) 26.10 -t 1.70' (4) 20,100 i 3,400 1.36 ? 0.110 (10) 4.10 ? 0.450 (10) 21.80 ? 0.440 (10) 220,000 2 9,100 (10) 61,700 ? 1,300 (6) (10) (8)" 32,600 2 2,600 "Average 2 standard error of the mean. "Number of determinations in parentheses. the regenerating epidermis incubated alone a t ACKNOWLEDGMENTS 26°C or 37"C, but the incorporation of ("H)orotic acid into RNA is still higher in epidermis This investigation was supported by NIH incubated under conditions in which RNA is grant AM18219. lost. Therefore, at least a portion of the inThe authors wish to thank Anthony Brigandi creased specific activity of RNA is not simply for his expert help in this investigation. due to the loss of unlabelled RNA. The effects of 3-hour incubation on normal epidermis was studied only for normal epiLITERATURE CITED dermis attached to its dermis a t 26"C, in either Argyris, B.F. (1977) Suppressor cells in the spleen of tumorSTKM or RPMI, because these were the condiallosensitized mice. Cancer Res., 37t3390-3399. tions which gave the best preservation of R.NA Argyris, T.S. (1978) Epidermal growth and ribosomal RNA accumulation in regenerating mouse epidermis following in regenerating epidermis. Normal epidermis abrasion. J. Invest. Dermatol., 70:267-271. attached to its dermis, incubated a t 26°C in Argyris, T.S. (1977) Kinetics of regression of epidermal either STKM or RPMI, shows no loss of RNA hyperplasia in the skin of mice following abrasion. Am. J. Pathol., 88,575582. and DNA, and no significant change in the amount of acid soluble material. There is also Argyris, T.S. (1976) Unbalanced RNA accumulation in regenerating mouse epidermis following abrasion. J. Invest. good preservation of histological detail. IncorDermatol., 67: 7 18-722. poration of ("1 orotic acid into RNA is higher DeLapp, N.W., and M.A. Karasek (1976)Importance of pyin the RPMI medium than in buffered sucrose, rimidine nucleotide salvage pathways for DNA synthesis in skin. J. Invest. Dermatol., 66:30&312. but the latter is quite adequate. L.M., T.S. Argyris, and G.B. Gordon (1977) EpiIn conclusion, this study demonstrates that DeYoung, dermal ribosome accumulation during two-stage skin when studying RNA metabolism of mouse epitumorigenesis. Cancer Res., 37:38&393. dermis in vitro, it is important to determine the Flamm, W.G., M.R. Barnejee, and W.B. Counts (1966) Topical application of actinomycin D on mouse skin: Effect of effects of the incubation period on the RNA the synthesis of ribonucleic acid and protein. Cancer Res., content. Our study demonstrates that the RNA 26: 134S1360. content can significantly decrease, even within Mueller, S.N., and T.S. Argyris (1977) Patterns of growth and ribosome accumulation during 3-methylcholana 3-hour period, under certain in vitro condithrene-induced epidermal hyperplasia. Cancer Res., tions, which show little change in DNA con37t3400-3405. tent, good incorporation of (3H)orotic acid into Mueller, S.N., and T.S. Argyris (1975) Free and membraneRNA, and very good preservation of epidermal bound ribosomes in normal and methylcholanthrenetreated mouse epidermis. Lab Invest., 32.209-216. histological details.