[1] 178w Table 1. Effect of immunoglobulin G (IgG) from unimmunized animals (designated preimmune IgG), MC-IgG, and PB-IgG on AHH activity of human monocytes and lymphocytes. AHH activity* (percent of control)t Donor/additions Monocytes Lymphocytes Uninduced BA-induced Uninduced BA-induced Donor 1 None 0.51 ± 0.12 (100) 5.70 ± 0.72 (100) Preimmune IgG 0.55 ± 0.04(108) 6.24 ± 0.33 (109) MC-IgG 0.24 ± 0.04 (47) 3.54 ± 0.43 (62) Donor 2 None 1.29 ± 0.18 (100) 18.78 ± 5.21 (100) 0.38 ± 0.08 (100) 1.33 ± 0.17 (100) Preimmune IgG 0.26 ± 0.06 (68) 0.83 ± 0.03 (62) MC-IgG 0.43 ± 0.15 (33) 7.97 ± 2.54 (42) 0.06 ± 0.04 (16) 0.17 j 0.04 (13) PB-IgG 1.52 ± 0.04 (118) 13.50 ± 2.12 (72) 0.18 ± 0.01 (47) 0.90 0.06 (68) Donor 3 None 0.35 ± 0.11 (100) 7.85 ± 2.21 (100) 0.47 ± 0.12 (100) 1.38 ± 0.06 (100) MC-IgG 0.10 0.05 (29) 2.72 ± 0.87 (35) 0.03 ± 0.03 (6) 0.15 ± 0.05 (11) PB-IgG 0.42 ±'0.11 (120) 7.60 ± 1.24 (97) 0.63 ± 0.16 (134) 1.09 ± 0.00 (79)

[2] 53w *AHH activity is expressed as units per miligam of protein; values represent the means ± standard deviation (four to eight determinations) except that where PB-IgG was used only two to four de n were made. tlTe activity of each cell sample in the absence of IgO (no additions) is designated as 100 percent.

[3] 13w The other numbers in parentheses show AHH activity in compaison to the control.

[4] 12w Aryl Hydrocarbon Hydroxylase Is Inhibited by Antibody to Rat Liver Cytochrome P450

[5] 200w Abstract. Antibody to the major purified cytochrome P450 induced by 3-methyl- cholanthrene in rat liver strongly inhibits aryl hydrocarbon hydroxylase activity of uninduced and benz[a]anthracene-induced human monocytes and lymphocytes. Antibody to the cytochrome P-450 induced by phenobarbital has relatively little or no effect on the aryl hydrocarbon hydroxylase activity of the same human cells. (NADH). The incubation for the AHH assay was started immediately after ad- dition of 100 nmole of BP in 50 A1 of absolute methanol and was continued in a water bath at 37°C with shaking under yellow light. After 30 minutes, the reac- tion was stopped by addition of 1 ml of cold acetone, and samples were process- ed ( 15). Both BP and BP metabolites were extracted with 3 ml of hexane, and the phenols were subsequently extracted with 1 ml of IN NaOH. Fluorescence was then determined (Aminco SPF 500 spectrofluorometer; excitation wave- length, 392 nm with a 5-nm bandpass; emission wavelength, 522 nm with a 10- nm bandpass). The AHH activity was calculated from a standard curve of 3-hydroxybenzo[a]pyrene (3-OH-BP), with 1 unit being defined as that amount of enzyme producing fluorescence equivalent to that of 1 pmole of 3-OH-BP per min- ute.

[6] 181w The effect of antibody on AHH activi- ty in untreated and benz[a]anthracene (BA)-treated monocytes and lympho- cytes from three individuals is shown in Table 1. Uninduced and BA-induced monocyte AHH activity was not sig- nificantly affected by incubation with IgG from animals prior to immunization or by PB-IgG (P > .05). However, the AHH activity of these cells was very signifi- cantly inhibited by MC-IgG (P < .005 in all cases). The enzyme activity in the presence of MC-IgG was 29 to 62 per- cent of the activity in the absence of anti- body. Lymphocyte AHH activity was sometimes inhibited by the PB-IgG (donor 2: uninduced, P < .005; BA-induced, P < .01; donor 3: uninduced, not signifi- cant; BA-induced, P < .005), but this in- hibition was not significantly different from that obtained with IgG from ani- monocytes and lymphocytes, the extent of inhibition was relatively independent of the cell concentration used. Thus, our results suggest either different sets of cytochrome P-450's in monocytes and lymphocytes or possibly different acces- sibility of the enzyme to antibody in the different cells.

[7] 42w It has been proposed that the susceptibility of individuals to the development of cancer may be related to their ability to metabolize carcinogenic compounds (16). This capacity is under some degree of genetic control (15,17), at least part of 0.4 0.6 0.8

[8] 261w which may be reflected in the proportions of the various cytochromes P-450 in the target tissue. Our results open a nphocytes by MC-IgG as a new approach to the examination of hu- man AHH, and may be helpful in deter- mining the heterogeneity of cytochrome P-450 in different tissues of different indimals prior to immnunization. The MC-IgG reduced AHH activity of uninduced and BA-induced lymphocytes to 7 to 16 per- cent of the control and this inhibition was well above that observed in these cells with IgG from animals prior to immunization (P < .0005 in all cases). Sensitivity of the AHH activity of mono- cytes and lymphocytes to the antibody preparations was unaltered by BA treat- ment of the cells. In rat liver microsomes the sensitivity of AHH activity to the antibodies is enhanced by polycyclic hydrocarbon treatment (13). This suggests that the polycyclic hydrocarbon-induced cytochrome P-450 in liver is different from the major form present in un- induced liver. In contrast, the AHH of control and BA-induced monocytes and lymphocytes is similarly inhibited by the antibody preparations, although the AHH of the cells is elevated by BA treatment. This suggests that in human mono- cytes and lymphocytes the cytochrome P-450 forms induced by BA treatment are the same as the forms in the un- treated cells. Our results also show that the cytochrome P-450 of both uninduced and BA-induced human monocytes and lymphocytes is antigenically similar to the major cytochrome P-450 form pres- ent in microsomes from MC-treated rat liver and relatively unrelated to the phe- nobarbital-induced liver cytochrome.

[9] 58w The AHH activity of lymphocytes was more strongly inhibited by MC-IgG than that of the monocytes (Fig. 1). With monocytes (Fig. lA), inhibition of activity increased gradually with increasing antibody concentration and reached 50 percent at about 0.4 mg of MC-IgG per milliliter, whereas with lymphocytes (Fig. IB) we observed an 80 percent inhi- bition at only 0.

[10] 60w 1 img/ml. With both viduals. This assessment may lead to an understanding of the relation of mixed function oxidase profile and susceptibility to chemical carcinogenesis. K. ROBIE-SUH R. ROBINSON H. V. GELBOIN Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20205 F. PETER GUENGERICH Department ofBiochemistry and Center in Environmental Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

[11] 102w Genetic manipulation of mammalian cells growing in tissue culture has been accomplished by various methods (1). Recently we described a technique for inserting genes for drug resistance into cells of living mice (2). In this procedure, calcium phosphate precipitates of mouse DNA containing multiple copies of the gene for dihydrofolate reductase (DHFR) were used to transform bone marrow cells to a state of increased resistance to the drug methotrexate. Transformed cells had a proliferative ad- vantage over untransformed cells under the selective pressure of methotrexate administration. Ultimately, the trans- formed methotrexate-resistant cells dominated the proliferating hemato- poietic cell population of the mice.

[12] 64w Two obvious potential uses of this gene-insertion technique are apparent for use in man: (i) induction of a higher degree of bone marrow resistance to the toxicity of anticancer drugs like metho- trexate and (ii) the introduction of a new gene that, by itself, does not confer any proliferative advantage on recipient cells, by linking it to a second gene con- ferring drug resistance.

[13] 86w We set out to devise conditions in which we could use the herpes simplex virus (HSV) thymidine kinase (tk) gene as a selective marker even in tk+ cells, which have the normal mammalian tk gene. We reasoned that use of appropriate levels of methotrexate and exoge- nous thymidine should cause cell growth to be limited by the thymidine kinase produced by the normal mammalian tk gene. Consequently, transformants car- SCIENCE, VOL. 208, 30 MAY 1960 and L. Ernster,Biochim. Biophys. Acta 473, 149 (1978). 17 Genet. 39,561(1978).

[14] 18w 18. Suported in part by PHS pant ES 00267 and 0190, NC contract NO1 CP 85s2 (NCI); and

[15] 73w (to F.P.G.) PHS research career development award ES 00041. We thank Drs. A. C. Peacock and E. Gozukara of the NCI for reviewing this report duing its preparation. 30 July 1979; revised 28 November 1979 rying additional tk genes would have a selective advantage. Moreover, we noted that the HSVtk enzyme has a greater affinity for its substrate than does the normal mammalian enzyme and that this might confer an additional selective advantage.

[16] 36w We now describe our success in first transforming tk+ mammalian tissue cul- ture cells'to drug resistance with herpes- virus tk gene in vitro and then selecting, in intact animals, bone marrow cells transformed with HSVtk gene.

[17] 8w We used the technique of Wigler et al.

[18] 96w Table 1. Thymidine kinase specific activity of mouse L cells transformed in vitro with HSVtk DNA. Clones were derived from the parent tk-L cell line or tk+ wild-type L cells (NCTC) by transformation with calcium-precipitated HSVtk DNA (1), with 4 to 20 ,ug of tk DNA. The tk DNA was either in circular form in plasmid pBR322 or in plasmid that had been cut with sal and ligated (9). aones of Ltk-lineage were selected in HAT medium. Cells of NCIC (wild-type) lineage were se- lected for in medium containing 10-4M metho- trexate and thymidine (0.5 ,ug/ml).

[19] 15w lineage Untransformed Ltk-Ltk+ (wild type) 207 205 214 Transformed with HSVtk in phc Ltk-Ltk+ Ltk+

[20] 100w Ltk+ (1) to insert the herpesvirus tk gene (3) in tissue culture cells. Mouse L cells lacking thymidine kinase (tk-) were incu- bated with a calcium phosphate precipitate of DNA containing the herpes tk gene in the plasmid vector pBR322 and then transferred to selective HAT medium (hypoxanthine, aminopterin, thymidine) (1). The anticipated results for transformation of Ltk-cells to tk+ status were observed, and-transformation efficiency was approximately the same as that reported by Wigler et al. ( 1) (Table 1). Thymidine kinase specific activity of different clones varied from 1.8 to 4.2 times the specific activity of wild-type (tk+) cells.

[21] 99w In addition to transforming tk-cells to tk+ status, we wished to transform wildtype (tk+) cells so that they would con- tain additional copies of the tk gene of viral origin. Wild-type (tk+) cells will grow in HAT medium by utilizing the available thymidine. Consequently, we explored a range of methotrexate and thymidine concentrations that would in- hibit normal cells with a single copy of tk gene but allow cells with increased concentrations of tk to grow. These se- lective conditions required higher folate antagonist concentrations (methotrexate, 10-4M) and lower thymidine levels (0.5 ,ug/ml) than used in conventional HAT medium.

[22] 101w Wild-type (tk+) L cells were exposed to herpesvirus tk DNA-under trans- forming conditions and then cultured un- der selective conditions. A number of transformed clones were isolated, and some were grown to sufficient density to allow for measurement of thymidine ki- nase specific activity (Table 1) and for analysis of tk-specific gene sequences in DNA, which we identified by Southern (4) hybridization (data not shown). Transformation efficiency of tk+-i tk++ varied between 0.2 and 5 colonies per 106 cells when 4 Ag of HSVtk DNA was used. Spontaneous growth of tk+ cells in the selective medium was < I per 108.

[1] 204w om Day et al. (8) we k. = 1.1 x 1011 molecuk cm-3, with a probable uncertainty ofabout ai fa- tor of 2. Based on this, our value for the inter- cept k1 (0,017 sec-') leads to a value for k,of 1.5 x 13 cm' molecule' sec-, lower by a factor of about 7 than Calear's vale (9). From the observed dependence on (CO] and [O21, we obtain k/1 6.3 + 0.2 x 10' second, from which a value of 1.4 x 10-'8 cm' mole- Human monocytes and lymphocytes metabolize the carcinogenic polycyclic hydrocarbon benzo[a]pyrene (BP) (1). This activity is mediated by aryl hydrocarbon hydroxylase (AHH), a cytochrome P-450-dependent mixed function oxidase system in mammalian liver (2, 3), lung (3,4), and other tissues (3,5). The AHH activity is induced in mammalian liver by a wide variety of xenobiotics, including polycyclic hydrocarbons and phenobarbital. Induction in liver by cule' secI can be derived for kC. his is five odersiof magnitde lower tha Callears valu (9), whih must be in efi as a reult of th coMPlexity of tc kinfic drivatio hisn Ns sh dealin th m. p eld surficc _ d of ord n that we are d4 ih h mechnsm at these lw concentrations.

[2] 12w 11. K. Poiter and D. H. Volman, Anal. Chem. 34, 748 (1962).

[3] 39w 12. In our experience CO cycliaden contain vari- able amount of metal carbonyts, usually iron, up to about 100 ppm. This is rmoved by pas- sage over an iodied-charcoal trp at face ve- locits as fast as 20 sec-'.

[4] 247w 13. F. W. S , Jr., personal commu tion. In our calultons we assume in s mixig and no eect on the lifetime aising from the presec of the rats. Both assumptons are subject to error. 14. We wish to acknowled the able assitane of B. Walunas and the suppot of the Depart- meat of Buergy under contract EE-77-S-02-4499. 27 September 1979; revised 19 February 1980 polycyclic hydrocarbons results also in the appearance of a form of the cyto- chrome different from the major form present in the untreated animal. This new form of cytochrome P4SO exhibits different substrate specificity and has dif- ferent susceptibility to the inhibitor 7,8- benzoflavone than the uninduced en- zyme (6). Treatment with phenobarbital causes an increase in another form of cytochrome P450 (7). Several of these cytochromes P450 have been purified from rat (8,9), rabbit (10), and 'ouse (11) liver. The AHH of human mnonom cytes and lymphocytes is induced by polycylic hydrocarbons in cell cuitUr . (12). However, since AHH in these cells is low and only very small amounts of cells from individual donors are avail- able, direct studies of cytochrome P-450 multiplicity in monocytes and lymphocytes have not been reported. In this study rabbit antibodies (IgG, iinmuno- globulin G) to the major purified forms of cytochrome P-450 from rats treated with 3-methylcholanthrene (MC-IgG) and from rats treated with phenobarbital (PB-lgG) (8, 13) were used to investigate the nature and diversity of AHH activity of human monocytes and lymphocytes.

[5] 148w Monocytes and lymphocytes were iso- lated from leukocyte-rich plateletpheresis residues obtained from blood of normal volunteers, cultured, and harvested as described previously (14, IS). For inhibi- tion experiments, monocytes (2.5 x 10" to 5 x 106 cells) and lymphocytes (5 x 106 to 10 x 106 cells) were first incu- bated at room temperature in 0.5 ml of 50 mM potassium phosphate buffer (pH 7.55) either alone or with-Ig from rabbits prior to imm tion, MC-IgG, or PB-IgG. Antibody was used At 1 mg per 0.5 ml, except where indicated other- wise. After the preliminary incubation, 0.5 ml of solution containing the remaining assay, ingredients was added to give final concentrations of: 50 mM phosphate buffer, pH 7.55, 4 mM MgC12, 25 mM nicotinamide, 0.7 mg of bovine serum albumin per milliliter, 0.8 mhM reduced nicotinamide adenine dinucleotide phosphate (NADPH), and 1.0 mM re- duced nicotinamide adenine dinucleotide

[6] 74w On the basis of the success of selecting Specific for cells transformed to tk++ status in vi- activity tro, we undertook parallel experiments (cpm/pAg) in mice. Our strategy was similar to that employed for selection of expression of 0.08 the DHFR gene (2). Mouse bone marrow 4.9 to 5.5 cells with a distinctive chromosomal marker (T6T6) were obtained from intact asmid normal animals and treated in vitro with 19.9 a calcium microprecipitate of herpes-

[7] 118w virus tk gene (3,5). The treated marrow, presumed to contain a few stem cells 22 transformed by viral gene, was mixed in a ratio of 1:1.5 with ""mock" trans- Insertion of a New Gene of Viral Origin into Bone Marrow Cells of Mice Abstract. DNA containing the herpes simplex virus thymidine kinase (HSVtk) gene was used to transform wild-type tk+ mouse L cells to a tk++ status in vitro using methotrexate as a selective agent. HSVtk DNA was also used to transform mouse bone marrow cells in vitro. Transformed marrow cells injected into irradiated and methotrexate-treated recipient mice gave rise to proliferating cells which in some cases dominated the marrow population and which contained HSVtk gene se- quences.

[8] 5w 00365/80/O530-1033$O0.50/0 Copyright C 1980 AAAS