Organ-specific effects of DNA methylation by alkylating agents in the inbred Swiss mouse.

Young adult inbred Swiss mice given single or repeated equitoxic doses of N-methyl-N-nitrosourea (MNUA) or methyl methanesulphonate (MMS) develop thymomas and pulmonary adenomas only following MNUA in spite of nearly identical overall alkylation of DNA of tumour target tissues by both agents due mainly to the biologically ineffective product 7-methylguanine. The main difference in DNA alkylation was the production of O6-methylguinine, a known pre-mutagenic product, by MNUA in amounts 10 or more times as large as following MMS. This supports the possibility that somatic mutations are a part of the process of carcinogenesis.     

Chromosome damage in the bone marrow of mice treated with the methylating agents methyl methanesulphonate and N-methyl-N-nitrosourea in the presence or absence of caffeine, and its relationship with thymoma induction.

A single dose (0.8 mmole/kg) of N-methyl-N-nitrosourea (MNUA) causes significantly more chromosome damage in the bone marrow of mice than a dose of equal toxicity to the animals, (1.1 mmole/kg) of methyl methanesulphonate (MMS) 6, 24 and 48 h after treatment. At these doses both agents alkylate bone-marrow DNA to similar extents, but only MNUA induces thymic lymphomata. The greater chromosome-damaging effects of MNUA are ascribed to the known differences in the pattern of DNA alkylation by each agent, in particular the much higher levels of O-6 methylguanine and phosphotriesters produced by MNUA. The greater chromosome-damaging effect of MNUA may account for its higher toxicity to the bone marrow which in turn may be a significant factor in the induction of thymomata. The enhancement by caffeine of chromosome damage seen particularly 48 h after MMS-treatment suggests that post-replication repair protects cells from the effects of DNA-methylation in vivo.     

Use of UV endonuclease from Micrococcus luteus to monitor the progress of DNA repair in UV-irradiated human cells

A sensitive enzymatic assay has been developed to follow the progress of NDA repair in human cells exposed to UV radiation. The assay employs an endonuclease selectively active at sites containing pyrimidine dimers in UV-damaged DNA. Primary fibroblasts are exposed to 254 nm radiation and incubated for specified times, their radioactivity labelled DNA is isolated and treated with a UV endonuclease extensively purified from Micrococcus luteus. Endonuclease-susceptible site remaining in the DNA are subsequently observed as single-strand scissions by sedimentation in alkaline sucrose gradients. In comparison to the situation with excision-proficient normal cells, those derived from patients suffering from either the classical or the De Sanctis-Cacchione clinical form of Xeroderma pigmentosum (XP) exhibit a marked diminution in the rate of disappearance of nuclease-susceptible lesions with time of post-UV incubation.     

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.     

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested).The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments.Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity.These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.     

Ethylation pf DNA and protamine by ethyl methanesulfonate in the germ cells of male mice and the relevancy of these molecular targets to the induction of dominant lethals

The molecular dosimetry of ethyl methanesulfonate (EMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 200 mg/kg of [³H]EMS and the ethylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 2-week period. The ethylations per sperm head closely paralleled the dominant-lethal frequency curve for EMS, reaching a maximum of 5 to 6.5 million ethylations per vas sperm head at 8 to 10 days after treatment. Ethylation of sperm DNA was greatest at 4 h after treatment, with 5.7 ethylations/10⁵ deoxynucleotides, and gradually decreased to 2.2 ethylations/10⁵ deoxynucleotides at 15 days after treatment. The ethylation of sperm DNA did not increase in the germ-cell stages most sensitive to EMS, ans was not correlated with the dominant-lethal frequency curve for EMS. However, ethylation of sperm protamine did increase in the germ-cell stages most sensitive to EMS, and showed an excellent correlation with the incidence of dominant lethals produced by EMS in the germ cells.A model is presented to explain, at a molecular level, how dominant lethals may be induced in mouse germ cells by EMS. Ethylation by cysteine sulfhydryl groups contained in mouse-sperm protamine could block normal disulfidebond formation, preventing proper chromatin condensation in the sperm nucleus. Stresses in the chromatin structure could then eventually lead to chromosome breakage, with resultant dominant lethality.     

Enhancement by alkylating agents of chemical carcinogen transformation of hamster cells in culture

When Syrian hamster embryo cells were pretreated with a weak chemical carcinogen, methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS), or with a physical agent such as X-irradiation prior to being exposed to a potent cancer-producing chemical, transformation (crisscrossing of cells not seen in control) occurred up to nine times more often than when the cells were not pretreated. The degree of enhancement appears independent of carcinogen dose. The transformation frequency associated with the carcinogens benzo(a)pyrene (BP), dimethylbenz(a)anthracene (DMBA), 3-methylcholanthrene (MCA), N-acetoxy-2-acetylaminofluorene (AcAAF), and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was increased. There are similarities in the enhancement produced by pretreatment of hamster cells with X-irradiation and with alkylating agents: with both, maximum enhancement occurred approx. 48 h after treatment and lethality attributable to the pretreatment was 10–20% relative to control. However, enhancement produced by X-irradiation pretreatment was slightly greater than that obtained with MMS. The exact cause of the enhancement in transformation resulting from the interaction of these agents is not yet known, but the enhancement associated with MMS pretreatment cannot be related to partial cell synchronization or disruption in the cell cycle. Hamster cells pretreated with 250 μM of MMS demonstrated no alteration in normal cel DNA synthesis through 48-h post-treatment. Analysis of unscheduled DNA synthesis by autoradiography or by alkaline sucrose gradients indicated that the damaged DNA was rapidly repaired after treatment. Therefore, repair of DNA damage as it is now understood is probably not involved.     

Dosimetry studies on the ethylation of mouse sperm DNA after in vivo exposure to (3H)ethyl metanesulfonate

Methods for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of mouse spermatozoa in the vasa deferentia and epididymides have been developed. These include procedures for the removal of contaminating protamine, which, like DNA, possesses nucleophilic sites that can be ethylated by EMS. At least 99% of all sperm protamine (at a 95% confidence level), as well as any other cellular contaminants, is removed during purification of the DNA. The purified DNA recovered from spermatozoa gives no indication of a preferential recovery of either (G+C)-rich or (A+T)-rich regions of the mouse genome: the [¹⁴C]dT/[³H]dC ratios for whole sperm and sperm DNA were the same for each animal tested.The spermatozoa of males used in the dosimetry studies were labeled with [¹⁴C]thymidine, and then the animals were given various [³H]EMS doses intraperitoneally. A constant exposure time of 4 h was used. The ratios of ³H and ¹⁴C activities in whole sperm and purified sperm DNA were used to measure the percentage of the total sperm ethylation occurring in the DNA. The maximum percentage found was about 18% in the dose range of 100–400 mg/kg. Values for the ethylations per nucleotide (E/N) ranged from ～ 10^?7 at 3.3 mg/kg up to ～ 10^?4 at 400 mg/kg, and the data indicated that E/N increased with the 1.5 power of the dose. E/N was also measured in testicular DNA, and the values obtained were close to those found for spermatozoan DNA.The results of such chemical dosimetry studies will be far-reaching in the interpretation of molecular events responsible for genetic alterations. As an example, dominant lethal studies by others, using EMS in the dose range considered in the present paper, have shown little or no effect until two or more days after injection of the mutagen into male mice. Since many sperm DNA ethylations are found after a 4-h exposure to EMS it appears that most of these DNA ethylations are not genetically important, at least in the production of dominant lethals, and that perhaps genetic damage occurs only at rarely ethylated DNA sites.     

Repair of DNA damage induced by methylating agents in human uterine cervical cells with or without cancer precursor lesions

Experiments were carried out to study the repair capabilities of normal human cervical fibroblasts and fibroblasts derived from human uterine cervical dysplasia, carcinoma in situ and invasive carcinoma. Sedimentation analysis of DNA in alkaline sucrose density gradient was carried out to monitor the DNA damage induced by a methylating carcinogen, methylnitrosourea (MNU). The results indicate that none of the cell lines, namely, fibroblasts either derived from normal human uterine cervix (T₃₀-11) or from cervical cells of cancer precursor lesions (T₄-3F; T₂₃-3; T₁₈) exhibited any significant repair in 72 h. In contrast fibroblasts derived from normal human skin (GM105) exhibited 38% repair of their DNA damaged by MNU. Epithelial-like cells (T₄-3E) obtained from cervical dysplasia exhibited only 18% repair of MNU-induced DNA damage in 72 h.When the damage was induced by another methylating agent, methyl methanesulfonate (MMS), fibroblasts from normal human skin (GM105) exhibited 40% repair of the damaged DNA whereas fibroblasts from normal human uterine cervix (T₃₀-11) exhibited only a 16% repair, in 72 h.These results suggest that fibroblasts derived from either normal human uterine cervix or from cervix with cancer precursor or cancer lesions exhibit low levels of repair of DNA damged by methylating agents.     

Identification of N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,N-dimethylnitrosamine or 1,2-dimethylhydrazine

Human Tamm-Horsfall urinary glycoprotein from an individual of the blood group Sd(a+) phenotype was tritium-labelled by treatment with galactose oxidase and sodium boro[3H]hydride and was then digested with endo-beta-galactosidase. A series of dialysable, labelled fragments was released from which a pentasaccharide was isolated that strongly inhibited the agglutination of Sd(a+) red cells by human anti-Sda serum and hence contained the Sda determinant structure. Reduction, methylation analysis and sequential exo-glycosidase digestion established the structure of the pentasaccharide as: GalNAc beta(1 leads to 4)[NeuAc(2 leads to 3)]Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3)Gal     

DNA modification in rat lungs following intratracheal or subcutaneous administration of 4-nitroquinoline-1-oxide, benzo[a]pyrene or 2-aminoanthracene

Benzo[a]pyrene (BP)-, 2-aminoanthracene (2AA)- and 4-nitroquinoline-1-oxide (4NQO)-mediated DNA modification were investigated in rat lungs by using alkaline sucrose gradient sedimentation. The exposure-route, the physicochemical nature of the administered compound and the number of treatments were all important in determining the extent of DNA modification. 4NQO produced qualitatively similar modification whether instilled intratracheally (i.t.) as a suspension or injected subcutaneously (s.c.) in a soluble form. BP and 2AA produced no DNA alteration when injected s.c; they did, however, modify DNA sedimentation when instilled as a suspension, but not until 24 h after treatment. Furthermore, BP caused no DNA modification at any sampling time when instilled in a lipid solvent. In contrast to the DNA modification observed at 24 h after a single i.t. treatment with a BP suspension, no such alteration was detected 12 or 24 h after the last of 5 similar daily treatments. These results are discussed with respect to mechanisms of differential transport, clearance and metabolism of administered carcinogens.     

The formation and repair of single-strand breaks in DNA of cultured mammalian cells treated with UV-light, methylating agents or 4-nitroquinoline-1-oxide.

The technique of sedimentation in alkaline sucrose was used to examine the formation and repair of single-strand (SS) breaks in cultured mammalian cells that were treated with methyl methanesulfonate (MMS), methyl nitrosourea (MNUA), 4-nitroquinoline-1-oxide (4NQO) or UV-light. The SS breaks induced by MMS and 4NQO were largely repaired by HeLa cells during a 5-h post-treatment incubation. The SS breaks induced by MNUA and UV-light were not repaired by HeLa cells. L-cells were not able to repair the SS breaks induced by any of the agents, which correlates with the deficiency of these cells for repair synthesis of DNA. The following conclusions are discussed. MNUA and UV-light produce modifications in DNA which are not repaired but are translated into SS breaks in alkali. MMS produces SS breaks intracellularly but these are not derived from a simple depurination of methylated purines. 4NQO produces a modification in DNA which is translated into an SS break in alkali but which can be removed by an intracellular process.     

Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice

The molecular dosimetry of methyl methanesulfonate (MMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 100 mg/kg of [3H]MMS and methylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 3-week period. The methylations per sperm head paralleled the dominant lethal frequency curve for MMS, reaching a maximum of between 22 and 26 million methylations per vas sperm head 8-11 days after treatment. Methylation of sperm DNA was greatest at 4 h (the earliest time point studied) after treatment, with 16.6 methylations/10(5) deoxynucleotides. DNA methylation gradually decreased during the subsequent 3-week period. The methylation of germ-cell DNA did not increase in the stages most sensitive to MMS (late spermatids leads to early spermatozoa) and was not correlated with the dominant lethal frequency curve for MMS. However, methylation of protamine did increase in the germ-cell stages most sensitive to MMS, and showed an excellent correlation with the incidence of dominant lethals produced by MMS in the different germ-cell stages. The pattern of alkylation produced by MMS in the developing germ-cell stages of the mouse is similar to that found for EMS. However, for equimolar exposures, MMS alkylates the germ cells 5-7 times more than does EMS. Hydrolyzed samples of protamine from [3H]MMS-exposed animals were subjected to thin-layer chromatography and amino acid analysis. Both procedures showed that most of the labeled material recovered from the hydrolysates co-chromatographed with authentic standards of S-methyl-L-cysteine. The amino acid analyses showed an average of approximately 80% of the labeled material eluting with S-methyl-L-cysteine. The mechanism of action of both MMS and EMS on the developing germ cells appears to be similar. The occurrence of S-methyl-L-cysteine as the major reaction product in sperm protamine after MMS exposure supports our initial model of how dominant lethals are induced in mouse germ cells by these chemicals: Alkylation of cysteine sulfhydryl groups contained in mouse-sperm protamine blocks normal disulfide-bond formation, preventing proper chromatin condensation in the sperm nucleus. Subsequent stresses produced in the chromatin structure eventually lead to chromosome breakage, with resultant dominant lethality.     

Variables affecting the selectivity and efficiency of retention of DNA fragments by E. coli RNA polymerase in the nitrocellulose-filter-binding assay

In this paper we characterize the effect of varying the solution conditions and filter-binding protocols on the extent and selectivity of DNA retention on nitrocellulose filters by DNA-binding proteins. These effects are illustrated by the binding interaction of Escherichia coli RNA polymerase with λ and T7 phage DNA restriction fragments. We present procedures which will help enhance the selective retention of some DNA restriction fragments over others. These include increasing the pH and salt concentration, decreasing the enzyme-to-DNA ratio, and including an appropriate washing step. Selective binding is not dependent on the presence of Mg²⁺. Although we only show data for RNA polymerase-DNA interactions, many of the principles discussed are likely to find practical applications in studying selective DNA-protein binding in general.     

The long N-terminus of the C. elegans DNA repair enzyme APN-1 targets the protein to the nucleus of a heterologous system

We previously isolated from a Caenorhabditis elegans cDNA library, designed for two-hybrid screening, a gene encoding the DNA repair enzyme APN-1 using cross-specie complementation analysis of the Saccharomyces cerevisiae apn1? apn2? tpp1? triple mutant deficient in the ability to repair several types of DNA lesions including apurinic/apyrimidinic (AP) sites. We subsequently purified the APN-1 from this yeast mutant and demonstrated that it possesses four distinct DNA repair activities. However, following the re-annotation of the C. elegans genome we discovered that the functionally active APN-1 encoded by the cDNA from the library might lack 108 amino acid residues from the N-terminus. We therefore synthesized the entire C. elegans apn-1 gene encoding the putative full-length APN-1 and created several N-terminal deletion mutants lacking either 63, 83 or 118 amino acid residues. The full-length APN-1, APN-1 (1–63Δ) and APN-1 (1–83Δ), but not APN-1 (1–118Δ) were stably expressed in the yeast triple mutant and cleaved the AP site substrate. However, only the full-length APN-1 rescued the yeast mutant from the genotoxicity caused by methyl methane sulfonate, a DNA damaging agent that creates AP sites in the genome. The full-length APN-1 was localized to the yeast nucleus, while APN-1 (1–63Δ) and APN-1 (1–83Δ) retained a cytoplasmic distribution. Our data suggest that the N-terminal region has no direct role in the DNA repair functions of APN-1 other than to target the protein to the nucleus and possibly to maintain its stability. Thus, the truncated APN-1, previously isolated from the two-hybrid library, ability to complement the yeast triple mutant depends on the engineered SV40 nuclear localization signal.     

The measurement of chemically-induced DNA repair synthesis in human cells by BND-cellulose chromatography.

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.     

Increased urinary excretion of nucleic acid and nicotinamide derivatives by rats after treatment with alkylating agents.

Rats treated with di(2-chloroethyl)methylamine (HN2), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine 0-24 h after treatment. Ethyl methanesulphonate (EMS) and dimethylnitrosamine (DMN) gave negative results in this respect but all five alkylating agents increased the excretion of 1-methyl-nicotinamide (1-meNmd). In addition, a larger quantity of 7-methylguanine (7MG) and uric acid was excreted after DMN treatment. 1,4-Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5-fluorouracil (5FU), cytosine arabinoside (araC), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz-[a]anthracene (7-BrMBA) gave negative results.     

Accumulation of short DNA fragments in hydroxyurea treated mouse L-cells

Treatment of L-cells with hydroxyurea markedly inhibits the incorporation of [³H]thymidine into DNA. The ³H incorporation that persists during hydroxyurea inhibition is largely into 7S DNA chains. The labelled fragments can be chased into higher MW DNA, suggesting that they are intermediates in the replication process. This interpretation concurs with that of earlier reports which describe a similar effect of hydroxyurea on the replication of viral DNA.     

The effect of growth state on the activity of the protein kinase isoenzymes an increase in type II cyclic AMP-dependent protein kinase is correlated with growth arrest in G1 phase

Native polyacrylamide gels have been used to resolve protein kinase isoenzymes from cultured cells and the protein kinases have been identified by carrying out phosphorylation reactions in the gel. Following electrophoresis, the gels were incubated with histome and [γ-³²P]ATP. The gels were then thoroughly washed and dried down, and the protein kinases were located by autoradiography. Protein kinase activity as measured in the gel system was a linear function of cytosol protein concentration up to about 100 μg per channel and incorporation of ³²P into histone was time dependent. Three bands of protein kinase activity were resolved in cytosol samples from baby hamster kidney (BHK) fibroblasts. The band with the lowest relative mobility utilized histone IIA or casein equally well as substrate protein whereas bands 2 and 3 demonstrated a clear preference for histone. Bands 2 and 3 displayed a relative mobility in electrophoresis that was identical to that observed for cyclic AMP-dependent protein kinases I and II from rat liver. Treatment of cyctosol samples with cyclic AMP prior to electrophoresis resulted in the disappearance of cyclic AMP-dependent protein kinases from the gel profile. This method was employed to identify bands 2 and 3 as cyclic AMP-dependent protein kinases. The protein kinases in growth-arrested cells were compared with proliferating cells. We have observed a 3.5-fold increase in the activity of Type II protein kinase as the cells arrest growth in G₁ phase of the cell cycle. This increase in Type II is correlated with the increase in cells blocked in G₁ and a decrease in II Type activity appears to be an early event in permitting cells to leave G₁ and resume growth.