Induction of erythroid differentiation in vitro by purines and purine analogues

Cell, Vol . 8, 263-269, June 1976, Copyright © 1976 by MIT

Induction of Erythroid Differentiation in Vitro by Purines and Purine Analogues

James F . Gusella Department of Medical Biophysics University of Toronto 500 Sherbourne Street Toronto, Ontario, M4X 1 K9, Canada David Housman Department of Biology and Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary The effectiveness of purines and purine analogues as inducers of erythroid differentiation in cultured murine erythroleukemia cells has been investigated . These cell lines have previously been shown to differentiate in vitro in response to dimethyl sulfoxide (DMSO) and a number of other polar solvents . Two purine analogues, 6-thioguanine and 6-mercaptopurine, as well as the naturally occurring purine, hypoxanthine, are shown to be extremely potent inducers . 6-Thioguanine is effective at a concentration of 0 .06 mM, 750 fold lower than the DMSO concentration required for equivalent induction . 6-Mercaptopurine and hypoxanthine are effective inducers at a concentration of approximately 2 mM . Accumulation of globin mRNA was monitored during induction with purine inducers and shown to be similar in amount to globin mRNA levels reached in DMSO-induced cultures . Induction of differentiation by all three compounds follows a similar time course to induction with DMSO . All three compounds are potent inducers of HGPRT (hypoxanthine-guanine phosphoribosyltransferase)-negative cell lines ; hence incorporation of purines into DNA is not required for induction of differentiation . Comparison of these compounds with other purines and purine analogues suggests a high degree of specificity in their interaction with a cellular target . Introduction Erythroleukemic cell lines derived from Friend virusinfected mice can be induced to undergo differentiation in vitro (Friend et al ., 1971) . In parallel with normal erythropoiesis, this process involves a series of coordinated biochemical events . Increased levels of heme synthesis (Friend et al ., 1971) and accumulation of globin mRNA (Ross, Ikawa, and Leder, 1972) lead to high levels of hemoglobin synthesis (Boyer et al ., 1972 ; Ostertag et al ., 1972) . Associated with induction of hemoglobin synthesis are alterations in the activities of enzymes of nucleotide biosynthesis which parallel quantitative changes

in enzyme activity observed during normal erythropoiesis (Reem and Friend, 1975) . The original report that dimethyl sulfoxide (DMSO) can induce differentiation has recently been complemented by reports from several laboratories that other organic compounds can induce this process . Tanaka et al . (1975) and Preisler and Lyman (1975) report that a series of highly polar compounds are inducers of erythroid differentiation for Friend cell line 745 . The effective range for the most potent of these compounds, 1-methyl-2-piperidone, was 3-10 mM . Leder and Leder (1975) report that butyric acid was an effective inducer of erythroid differentiation of another Friend cell line, T3CI2 . In this case, 1 mM was the minimum effective dose . During the course of selection of erythroleukemic cell lines resistant to 6-thioguanine, we observed that resistant cell lines were induced to differentiate in the presence of extremely low concentrations of 6-thioguanine . A dose of 0 .06 mM was effective in inducing cells to differentiate, a level 20 fold lower than that reported for butyric acid . Subsequent experiments revealed that 6-mercaptopurine and hypoxanthine were also effective inducers of erythroid differentiation of erythroleukemic cells . As a result of these observations, we undertook a quantitative characterization of the inductive effects of 6-thioguanine, 6-mercaptopurine, and hypoxanthine, as well as a systematic survey of the inductive capacity of compounds related to them in structure . Results Properties of Cell Lines Tested For the purpose of characterizing the inductive response to 6-thioguanine, three erythroleukemic cell lines, 745-TG-11, 745-TG-13, and Fsd-TG-6, were isolated by selection for resistance to 6thioguanine . 6-Thioguanine-resistant mutants of other cell lines are often deficient for the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) . This deficiency prevents growth in medium containing hypoxanthine, aminopterin, and thymidine (HAT medium) (Szybalski and Szybalska, 1962) . As shown in Table 1, the properties of the mutants we isolated are consistent with such a deficiency . Mutant clones plated with equal efficiency in the presence or absence of 0 .06 mM 6-thioguanine, while wild-type cell lines (745 and Fsd) were completely inhibited . Growth of mutant clones was completely inhibited in the presence of HAT, while the parent cell lines gave undiminished plating efficiencies . Direct assay of HGPRT activity revealed that all three mutant clones possessed <0 .1% of parental levels of the enzyme .



Cell 264

Table 1 . Properties of Cell Lines Tested HGPRT Activity pmoles/min/mg protein 745 745-TG-11 745-TG-13 FSD FSD-TG-6

Plating Efficiency Control

Thioguanine

6300

0 .33

<10-5

<6

0 .25

<6

0 .34

9700

0 .39

<6

0 .28

HAT

0 .19

<10 -5

0 .36

<10 -5

<10-5

0 .26

Inducible with DMSO

0 .32

+ +

+

0 .37 <10-5

+

HGPRT assays were performed as described in Experimental Procedures . Plating efficiency in 1 .4% methylcellulose was determined for the following : control (no additions), thioguanine (0 .06 mM), and HAT (10- 4 M hypoxanthine, 4 x 10 -7 M aminopterin, and 1 .6 x 10-5 M thymidine) .

Response Parameters for Induction of Differentiation by Purines To compare precisely the effectiveness of 6thioguanine, 6-mercaptopurine, and hypoxanthine with DMSO in the induction of differentiation of erythroleukemic cells, we performed two experiments, a dose-response determination and an analysis of the kinetics of appearance of hemecontaining cells . Initially, parallel cultures of 745-TG-1 1 were incubated with a range of concentrations of purine inducers or DMSO . The proportion of heme-containing cells as a function of dose is shown in Figure la . The relative effectiveness of each inducer is summarized in Table 2 . It is evident that 6-thioguanine has approximately 750 times the potency of DMSO on a molar basis . 6-Mercaptopurine and hypoxanthine were effective at a concentration range 30 fold higher than 6-thioguanine, but 25 fold lower than DMSO . As shown in Figure 1 b, the dose response of 745, the parent HGPRT-positive cell line, to hypoxanthine is very similar to that of the HGPRT-negative line 745-TG-11 . 6-Mercatopurine and 6-thioguanine cannot be tested with the wild-type cell line because of their toxicity. The appearance of differentiated cells following stimulation by DMSO requires a 48-72 hr latent period (Preisler and Giladi, 1975) . A comparison among inducers of the kinetics of appearance of differentiated cells was performed to assess the relative latent period for each inducer . The results are shown in Figure 2 . It is clear that all three purine inducers show similar induction kinetics and are indistinguishable from DMSO with respect to latent period . Measurement of Globin mRNA Levels A quantitative analysis of induction of erythroleukemia cells requires the measurement of at least two discrete parameters in the coordinated sequence of biochemical events associated with erythroid differentiation . Quantitation of a single parameter

Figure 1 . Dose Response to Purine Inducers (a) Cultures of 745-TG-11 were seeded at 5 x 10 4 cells per ml in a 35 mm petri dish (Falcon # 1008) in various concentrations of the indicated compounds and incubated for 6 days at 37°C . The proportion of differentiated cells was determined as described in Experimental Procedures . (o-o) 6-thioguanine : (o o) 6mercaptopurine ; (o A) hypoxanthine ; ( •-•) DMSO. (b) As in (a), except that the parental line 745 was used . 6Thioguanine and 6-mercaptopurine could not be tested due to toxicity .

Table 2 . Relative Potency of Purine Inducers Half-Maximal Dose (mM) Inducer

745

745-TG-1 1

DMSO

70

45

6-Thioguanine

NG

0 .06

6-Mercaptopurine

NG

1 .5

Hypoxanthine

2 .6

2 .0

NG designates no growth . Half-maximal dose is the dose causing half-maximal induction of differentiation (40% benzidine-positive cells) and was determined from the experiments shown in Figures 2a and 2b) .

such as levels of intracellular heme is not sufficient, since some compounds may exert a direct effect on one of the coordinated biochemical processes but not the others . For example, 1 mM S-aminolevulinic acid dramatically stimulates porphyrin synthesis in erythroleukemia cells while not altering levels of globin mRNA synthesis (A . Bernstein and D . Housman, unpublished observations) . Thus to quantitate further the inductive effect of purines on erythroleukemia cells, we chose to measure accu-



Purine Induction of Erythroid Differentiation 265

loo -(C3 )

_(b) + Hyponanthine + DMSO

-

•

-I Control

i 1 I I I I I 1 2 3 4 0 I 2 log RNA

3

Figure 2 . Time Course of Induction

Figure 4 . Globin mRNA Induction of 745

Cultures of 745-TG-11 were seeded at 5 x 104 cells per ml in the presence of the following : 210 mM DMSO (0-s) ; 0 .24 mM 6thioguanine (0-0) ; 2 .63 mM 6-mercaptopurine and 3 .67 mM hypoxanthine (A ) . An aliquot was removed at the indicated times from each of these, as well as from a control culture (c, A) which had received no additions . The proportion of differentiated cells was determined by benzidine staining as described in Experimental Procedures .

Cultures of 745 containing 210 mM DMSO (a) and 5 .8 mM hypoxanthine (b) were seeded at 5 x 104 cells per ml and incubated for 84 hr at 37°C . Cytoplasmic RNA was prepared and hybridized to mouse globin cDNA as described in Experimental Procedures . Each capillary contained 787 cpm of cDNA . A background value of 17 cpm was subtracted .

Control --, Control --., 20 _• - i I I I •- •' I I I I I 3 4 I 2 4 2 3 Micrograms RNA added Figure 3 . Globin mRNA Induction of 745-TG-11 Cultures of 745-TG-11 containing 2 .63 mM 6-mercaptopurine (a), 0 .24 mM 6-thioguanine (b), 3 .67 mM hypoxanthine (c), 210 mM DMSO (d), and no additions (control) were seeded at 5 x 104 cells per ml and incubated for 84 hr at 37°C . Cytoplasmic RNA was prepared and hybridized to mouse globin cDNA as described in Experimental Procedures . Each capillary contained 592 cpm of cDNA . A background value of 18 cpm (the value for a sample which received no RNA) was subtracted .

mulation of globin mRNA in the cytoplasm of induced cells . Cytoplasmic RNA was isolated from cells treated with inducers for 84 hr, a length of exposure sufficient to achieve maximal induction of globin mRNA with DMSO (Ross et al ., 1972) . Increasing amounts of cytoplasmic RNA were hybridized to a constant amount of radioactive DNA complementary to the mRNA synthesized in vitro

(cDNA) . Following hybridization, the proportion of cDNA in each sample in hybrid form was determined by digestion with the single-strand specific nuclease S1 . The results of a series of such experiments are shown in Figures 3 and 4 . All three purine inducers were tested with 745-TG-11, but only hypoxanthine could be tested with parent strain 745 . In all cases, a substantial increase ranging from 30 to 70 fold in globin mRNA levels was observed with purine inducers . These levels were comparable to increases in globin mRNA levels observed with DMSO . Like DMSO, all three purine inducers lead to globin mRNA levels which were increased at least 50 fold over levels for untreated cells . Independently Isolated Cell Lines Respond to Purine Inducers Induction of differentiation by purines could be a consequence of an alteration in purine metabolism peculiar to line 745 . To test this possibility, we measured the responsiveness to purines of an independently isolated erythroleukemic cell line, Fsd . Initial experiments were performed on this cell line measuring responsiveness to hypoxanthine . As shown in Table 3, Fsd is equally responsive to hypoxanthine and DMSO . To characterize further the inductive response of Fsd, a HGPRT-negative clone was isolated (Fsd-TG-6) and tested for responsiveness to 6-thioguanine and 6-mercaptopurine . As shown in Table 3, Fsd-TG-6 was as responsive to these compounds as 745-TG-11 . Thus induction of differentiation by 6-thioguanine, 6-mercaptopurine, and hypoxanthine appears to be a general property of murine erythroleukemia cell lines .



Cell 266

A Cell Line with a Preferential Response to Purine Inducers It would be of interest from both a genetic and biochemical point of view to isolate and characterize erythroleukemic cell lines which responded to purine inducers while not responding to DMSO . One such line was isolated during a selection for 6-thioguanine-resistant cells . As seen in Table 1, this cell line is deficient in HGPRT and shows equal sensitivity to HAT and resistance to 6-thioguanine compared to 745-TG-11 and Fsd-TG-6 . Analysis of the response of 745-TG-13 to DMSO and purine inducers (Table 3) clearly shows that 745-TG-13 does not differentiate when treated with 210 mM DMSO . In fact, higher doses up to and including a lethal dose of DMSO (350 mM) did not lead to any increase over background levels of differentiated cells . In contrast, treatment of 745-TG-13 with 6thioguanine (0 .24 mM), 6-mercaptopurine (2 .63 mM), or hypoxanthine (3 .67 mM) leads to a substantial increase in the proportion of differentiated cells .This result was confirmed and quantitated in a different manner by the isolation of cytoplasmic RNA from 745-TG-13 after treatment with DMSO (210 mM) and 6-thioguanine (0 .24 mM) . As shown in Figure 5, treatment of 745-TG-13 with 6thioguanine for 84 hr leads to a 10 fold increase in globin mRNA compared to an untreated culture . Levels of globin mRNA present in the cytoplasm of DMSO-treated and control 745-TG-13 cells were indistinguishable, and both were similar to levels found in untreated 745-TG-11 cells (Figure 3) .

One group of compounds which illustrates this interaction is the derivatives of 6-mercaptopurine (Table 4) . 6-Mercaptopurine induces differentiation with a half-maximal dose of 1 .5 mM (Figure 2) . Substitution of an NH 2 group in the 2 position of 6-mercaptopurine to yield 6-thioguanine increases the potency of the inducer about 20 fold . However, substitution of an OH (6-thioxanthine) or SH (2,6dithiopurine) at the 2 position abolishes the inductive effect . Modification of the 2-NH 2 group of thioguanine with an acetyl group (2-acetylamino6-mercaptopurine) decreases the inductive potency of the substituted derivative about 10 fold . Conversely, modification of the 6-SH group of thioguanine by the addition of a benzyl group gives a substituted derivative (2-amino-6 benzyl mercaptopurine) which is equally potent as an inducer . Derivatives of hypoxanthine, also shown in Table 4, illustrate a similar degree of specificity. As with 100

• + Thioquanine

Control

0

• +DMSO 20

0

I

I 6

5

µg

The Effects of Closely Related Compounds A comparison of purines which cause differentiation of erythroleukemic cells to other purines and purine analogues reveals a high degree of structural specificity in the inductive effect . In particular, the character of substituents at the 2 and 6 positions of the purine ring appears to be critical in determining the effectiveness of a purine in inducing differentiation .

-

RNA

Figure 5 . Differential Response of 745-TG-13 to DMSO and 6Thioguanine Cultures of 745-TG-13 were seeded at 5 x 104 cells per ml and incubated for 84 hr at 37°C . Cultures contained either 0 .24 mM 6-thioguanine, 210 mM DMSO, or no addition (control) . Hybridization was performed as described in Experimental Procedures . Each capillary contained 522 cpm of cDNA . A background value of 18 cpm was subtracted in each case .

Table 3 . Induction of Independently Derived HGPRT-Negative Cell Lines by Purines and DMSO Benzidine-Positive Cells (%) DMSO

6-Thioguanine

6-Mercaptopurine

Hypoxanthine

745

1 .4

86

NG

NG

89

745-TG-1 1

2 .3

82

82

76

79

Control

745-TG-1 3 FSD FSD-TG-6

30

25

23

<1 .0

1 .6

42

1 .2

NG

NG

51

4 .2

91

96

89

78

Cultures of all the indicated cell lines were incubated for 6 days in the presence of 210 mM DMSO, 0 .24 mM 6-thioguanine, 2 .63 mM 6-mercaptopurine, 3 .67 mM hypoxanthine . The proportion of differentiated cells was determined by benzidine staining as described in Experimental Procedures . NG designates no growth .

Purine Induction of Erythroid Differentiation 267

Table 4 . Inductive Capacity of Purines and Purine Analogues Purine Substituents (Position) Compound

6

2

Hypoxanthine

OH

H

Other

Concentrations Tested (mM)

Benzidine-Positive Cells (%)

5 .5

82

3 .7

83

1 .8

34

0 .59 1-Methylhypoxanthine

0

H

1 CH 3

8 .2

3 .3

77

2 .0

36

0 .66

6 .6

0 .33

2 .2

3 .3

3 .9

2 .9

1 .6

Xanthine

OH

OH

Uric Acid

OH

OH

Guanine

OH

NH2

0 .50

3,6

Isoguanine

NH 2

OH

1 .0

2 .0

2-Aminopurine

H

NH2

2 .2

2,0

Adenine

NH 2

H

1 .5

1 .6

2,6-Diaminopurine

NH 2

NH 2

0 .25

61

0 .05

11

8 OH

Allantoin 6-Mercaptopurine

6 .3 SH

H

1 .8

3 .5

73

2 .0

51

1 .0

17

0 .05

5 .8

2-Mercaptopurine

H

SH

2 .0

3 .8

2,6-Dithiopurine

SH

SH

2 .2

0 .6

6-Thioxanthine

SH

OH

1 .8

2 .4

2-Thioxanthine

OH

SH

1 .8

2 .2

6-Thiouric Acid

SH

OH

2 .7

2 .8

6-Thioguanine

SH

NH 2

8 OH

0 .24

82

0 .10

70

0,05

28

0 .01

6 .4

6-Amino-2-Mercaptopurine

NH Z

SH

2 .3

2-Amino-6-Benzyl-Mercaptopurine

SCH2O0

NH 2

0 .25

68

0 .17

52

0 .08

29

0 .04

10

2-Acetylamino-6-Mercaptopurine

SH

NHCOCH3

4 .8

2 .6

67

1 .5

38

0 .50

5 .6

0 .20

1 .2

Cultures of 745-TG-1 1 were incubated for 6 days in the presence of various purines and purine analogues, as well as allantoin, a nonpurine breakdown product of hypoxanthine and guanine . The proportion of benzidine-staining cells was determined as described in Experimental Procedures . In the case of a negative response (no significant induction), only the highest concentration tested is shown for that compound .

Cell 268

6-mercaptopurine, substitution of an OH (xanthine) or an SH group (2-thioxanthine) in the 2 position abolishes the inductive effect . In this case, however, substitution of an NH 2 group in the 2 position to give guanine did not increase the potency of the compound as an inducer . Unfortunately, guanine could not be tested above 0 .5 mM due to its insolubility . Methylation of the 1 position (1-methylhypoxanthine) of hypoxanthine does not affect induction . Another compound which was found to have significant inductive ability was 2,6-diaminopurine . However, neither 2-aminopurine nor 6-aminopurine showed a similar effect . Major products of catabolism of inductive purines are not inducers of differentiation . Thus xanthine, uric acid, and allantoin, direct breakdown products of hypoxanthine, are not effective inducers . Similarly, 6-thioxanthine and 6-thiouric acid, potential breakdown products of 6-thioguanine and 6-mercaptopurine, do not induce differentiation . Discussion Our results have shown that a number of purines serve as inducers of differentiation of erythroleukemic cells at very low molar concentrations . The observation that a naturally occurring molecule such as hypoxanthine and its direct analogues 6thioguanine and 6-mercaptopurine are inducers of erythroid differentiation raises a number of significant questions . One consideration raised by these observations is the use of thioguanine resistance and HAT resistance as selective methods in genetic studies of erythroleukemic cells. We have found that prolonged growth of erythroleukemic cells in the presence of an inducing agent can select for a nondifferentiating phenotype (J . Gusella and D . Housman, unpublished observations) . The observation that 6-thioguanine and hypoxanthine are potent inducing agents suggests that it is advisable to remove a selected clone from the presence of the inductive purine once the initial selection has been accomplished . This procedure was effective in maintaining a responsive state for all three thioguanine-resistant cell lines which we isolated . Induction of differentiation of erythroleukemic cells by purines may reflect a control mechanism which is operative in normal hematopoiesis as well . Reem and Friend (1975) have demonstrated alterations in levels of enzymes involved in purine metabolism following DMSO induction of erythroleukemic cells . The observed changes parallel those occurring during erythropoiesis in vivo . However, none of the observed changes in enzyme levels appears to have a direct bearing on the

metabolism of hypoxanthine, 6-thioguanine, and 6-mercaptopurine . In the experiments we have described, it is clear that incorporation of inductive purines into DNA or RNA is not required for induction to take place . We therefore suggest that inductive purines interact directly as the free base with a discrete cellular target . Identification and characterization of this cellular target would be extremely important in elucidating control of the differentiation process . The basic similarity in the response parameters of erythroleukemic cells to purine inducers and DMSO might indicate a common mechanism of induction by these compounds . However, the existence of a cell line such as 745-TG-13, which responds to purines but not to DMSO, may argue against this possibility . Since the mode of action has not been established for DMSO or any other inducer, comparisons at this level are not presently possible . Because cells are so highly permeable to DMSO, even the localization of a target to the cell surface or the interior of the cell has not been possible . Mutant mammalian cells have been described which are altered in the ability to transport purines (Harris and Whitmore, 1974) . The isolation of similar erythroleukemic cell lines would facilitate the localization of the target for induction of differentiation by purines. Experimental Procedures Culture Conditions Cultures were maintained in alpha medium (Stanners, Eliceiri, and Green, 1971) lacking nucleosides, supplemented with 15% fetal calf serum (Reheis Chemical Company, Phoenix, Arizona) . Determinations of cell number were made using an automatic cell counter (Coulter Counter Model ZB1 ; Coulter Electronics, Inc ., Hialeah, Florida) . Determination of plating efficiency was by culture in alpha medium lacking nucleosides, containing 1 .4% methylcellulose (Dow Chemical Company, Wellesley, Massachusetts) and 20% fetal calf serum . Cell Lines 745 was obtained from Dr. C . Friend and Fsd from Dr . D . Kabat . To mutagenize these lines, we treated exponentially growing cultures with 300 µg/ml of ethyl methane sulfonate (EMS) for 12 hr and allowed them to recover for 48 hr . Survival after EMS treatment was generally 10-20% . The cells were then plated in methylcellulose in the presence of the selective agent . 745-TG-1 1 was obtained by plating a mutagenized culture of 745 in 0 .06 mM 6-thioguanine . 745-TG-1 3 was cloned from an unmutagenized culture of 745 which was allowed to remain unfed in the presence of 0 .06 mM 6thioguanine for 3 weeks . Fsd-TG-6 was obtained by plating a mutagenized culture of Fsd in the presence of 0 .24 mM 6-thioguanine . Benzidine Staining 0 .1 ml of cell culture was combined with 0 .1 ml of fetal calf serum . The cells were then spun down onto a microscope slide using a Shandon-Eliot cytocentrifuge . The preparation was fixed in methanol for 30 sec and then stained as follows : 5 min in 3,3'-dimethoxybenzidine solution (a 1 % solution in methanol ; 5 min in hydrogen peroxide solution (10 parts 30% H202 to 110 parts 70% ethanol) ;

Purine Induction of Erythroid Differentiation 269

2 min in distilled water ; 5 min in Harris-modified Hematoxylin stain solution (Fisher Scientific Company) ; and 5 min under running tap water . Under these conditions, heme-containing cells stain orange, while cells lacking heme stain blue . A minimum of 500 cells per slide was counted in making determinations of the percentage of differentiated cells . HGPRT Activity Cell-free extracts were prepared from 108 cells by freeze-thawing, followed by centrifugation and dialysis as described by Reem and Friend, 1975 . HGPRT activity was assayed by measuring conversion of 14Chypoxanthine to 14C-IMP (inosine 5' monophosphate) . The reaction mixture of 100 pl contained 1 mM PRPP (5 phosphorylribose-1pyrophosphate, sodium salt), 5 MM MgC12, 10 mM Tris-HCI (pH 7 .5), 1 MM 74 C-hypoxanthine (1 .94 mCi/mmole), and 40-60 µg of sample protein . Incubations were carried out at 37°C, and the reaction was stopped by the addition of 10 µI of 0 .25 M EDTA containing 6 mg/ml IMP, followed by rapid freezing in an ethanol dry-ice bath . Under these conditions, the assay was linear for at least 30 min . In cases where no HGPRT activity was detectable in a sample, the sensitivity of the assay was increased by using a higher specific activity of 1 4C-hypoxanthine (48 .6 mCi/mmole) at a lower final concentration of 0 .04 mM . Conversion of 14 C-hypoxanthine to 14C-IMP was monitored by electrophoresis of the reaction mixture . 10-20 µl of the sample were spotted on Whatman 3 mm paper and subjected to electrophoresis for 30 min at 4000 V in 0 .05 M borate buffer (pH 9 .0) containing 1 mM EDTA. In cases where the lower concentration of 1 4C-hypoxanthine was used, 5 µl of cold hypoxanthine solution (2 .2 mM) were also spotted on the paper . The locations of hypoxanthine and IMP were detected under ultraviolet light, and radioactivity was determined using a Beckman LS-230 scintillation counter at an efficiency of 40% . Protein determinations on all samples were made according to the method of Lowry et al ., (1951) . Cytoplasmic RNA Extraction 107 cells were spun down at 200 x g for 5 min and lysed in 1 ml of the following salt solution : 0 .14 NaCl, 5 mM KCI, 1 .5 mM MgC12 , and 0 .19 Triton X-100 . Nuclei were removed by centrifugation at 500 x g for 5 min and 1 ml of 2 x SDS buffer [0 .2 M NaCl, 20 mM Tris-HCI (pH 7 .3), 2 mM EDTA, 1% sodium dodecylsulfate] was added to the cytoplasmic extract . The sample was then incubated at 37°C for 2 hr with 300 .8g/ml of proteinase K . The sample was extracted with an equal volume of phenol :chloroform :isoamyl alcohol (100 :100 :1), and the organic layer was removed . The aqueous layer was then reextracted twice with chloroform :isoamyl alcohol (100 :1) . The RNA was then precipitated with 3 vol of 100% ethanol at -20°C for a minimum of 1 hr and collected by centrifugation for 30 min at 1400 x g at 4°C . Traces of ethanol were removed by vacuum dessication, and the pellet was resuspended in 100 µl of water . The OD at 260 and 280 of a 1 :20 dilution was measured on a Gilford 2400-2 spectrophotometer . Hybridization with cDNA cDNA was synthesized from purified mouse globin mRNA with AMV RNA-dependent DNA polymerase using 3H-dCTP (24 .8 Ci/mmole) as previously described (Housman et al ., 1974) . Hybridization was performed in a sealed 5 µl capillary in 0 .2 M sodium phosphate (pH 6 .8), 0 .5% SDS for a period of 40 hr at 70°C . Samples were diluted into 2 ml of digestion buffer [0 .1 M sodium acetate (pH 4 .5), 10 µg/ml denatured calf thymus DNA, 1 mM ZnSO 4] and treated for 30 min at 45°C with S1 nuclease (400 units per ml) . Carrier yeast RNA was added to a final concentration of 50 ug/ml, and samples were precipitated by the addition of 1 ml of cold 30% TCA . Samples were collected by filtration under vacuum onto 24 mm glass fiber filters (Whatman GP/C) and washed with 5% TCA .

The filters were dried and counted in a Beckman LS-230 liquid scintillation counter . The proportion of the cDNA hybridized in each case was calculated by subtracting the value obtained for a cDNA sample which received no RNA input, dividing by the number of cpm in an undigested sample, and multiplying by 100 . The values for hybridization of a series of RNA concentrations for each sample are plotted on a linear scale as a saturation curve . The slope of the linear portion of each curve is considered to be directly proportional to the globin mRNA content of the sample . Sources of Materials 6-Thioguanine, 6-mercaptopurine, and hypoxanthine, as well as all other purines and purine analogues, were obtained from Sigma Chemical Company (St . Louis, Missouri) . 3 H-dCTP and 14 C-hypoxanthine were supplied by New England Nuclear (Boston, Massachusetts) . Proteinase K was purchased from E-M Laboratories (Elmsford, New York), and S1 nuclease was obtained from Miles Laboratories (Kankakee, Illinois) . AMV RNA-dependent DNA polymerase was a gift from Dr . J . Beard . Acknowledgments We would like to thank V . Crichley and G . Weeks for excellent technical assistance . This work was supported by grants from the National Cancer Institutes of Canada and the United States . J . G . was the recipient of a 1967 science scholarship from the National Research Council of Canada . Received November 28, 1975 ; revised February 16, 1976 References Boyer, S . H ., Wuu, K. D ., Noyes, A . N ., Yound, R ., Scher, W ., Friend, C ., Preisler, H . D ., and Bank, A . (1972) . Blood 40, 823-835 . Friend, C ., Scher, W ., Holland, J . G ., and Sato, T . (1971) . Proc . Nat . Acad . Sci . USA 68, 378-382 . Harris, J . F ., and Whitmore, G . F. (1974) . J . Cell Physiol . 83, 43-51 . Housman, D ., Skoultchi, A., Forget, B . G ., and Benz, E . J ., Jr . (1974) . Ann . N . Y . Acad . Sci . 241, 280-289 . Leder, A ., and Leder, P . (1975). Cell 5, 319-322 . Lowry, O . H ., Rosebrough, N . J ., Farr, A . L ., and Randall, R . J . (1951) . J . Biol . Chem . 193, 265 . Ostertag, W ., Melderis, H ., Steinheider, G ., Kluge, N ., and Dube, S . (1972) . Nature New Biol . 239, 231-234 . Preisler, H . D ., and Giladi, M . (1975) . J . Cell Physiol . 85, 537-546 . Preisler, H . D ., and Lyman, G . (1975) . Cell Differentiation 4, 179-185 . Reem, G . H ., and Friend, C . (1975) . Proc . Nat . Acad . Sci . USA 72,1630-1634 . Ross, J ., Ikawa, Y ., and Leder, P . (1972) . Proc . Nat . Acad . Sci . USA 69, 3620-3623 . Stanners, C . P ., Eliceiri, G . L ., and Green, H . (1971) . Nature New Biol . 230, 52-54 . Szybalski, W., and Szbalska, E . H . (1962) . Univ. Mich . Med . Bull . 28,277-293 . Tanaka, M ., Levy, J ., Terada M., Breslow, R ., Rifkind, R ., and Marks, P . (1975) . Proc . Nat . Acad . Sci. USA 72, 1003-1006 .