Lysosomal and non-lysosomal factors in chemically induced chromosome breakage

Experimental Cell Research 64 (1971) 209-217

LYSOSOMAL AND NON-LYSOSOMAL FACTORS IN CHEMICALLY INDUCED CHROMOSOME BREAKAGE M. M. COHEN’ and ROCHELLE HIRSCHHORN IDivision of Human Genetics, Department of Pediatrics, State University of New York at Buffalo, School of Medicine, and Buffalo Children’s Hospital, and =Department of Medicine, New York University School of Medicine, New York, N. Y. 10003, USA

SUMMARY The possibility that chemically-induced chromosomal damage is mediated through the mechanism of lysosomal labilization was investigated. The known chromosome breaking- agents, mitomycin C and streptonigrin, showed no effect on lysosomal membranes as evidenced by both enzymatic and previous electron microscopic studies [7]. The combination of these drugs with known lysosomal stabilizing agents reduced the frequency of chromosome damage somewhat. However, chromosome damage was also reduced by exposure of cells to heat or cold. The known lysosomal labilizers, vitamin A alcohol and acid, did not increase the frequency of chromosome breakage in peripheral lymphocytes. Therefore, these studies fail to support the hypothesis that the destruction of lysosomal membranes and the subsequent release of hydrolytic enzymes are instrumental in the induction of chromosomal damage by mitomycin C and streptonigrin.

That enzymatic digestion of DNA by DNAase may cause chromosome breakage has been suggested by several authors [ll-131. This hypothesis has been supported by recent studies demonstrating chromosome anomalies following the destabilization of lysosomal membranes with release of lysosomal enzymes (presumably, including DNAase) in human diploid cells [l] and following the injection of isogenic lysosomes into mice

[W. The following experiments were performed to examine further the possible role of lysosomes in chemically-induced chromosome breakage. It had been demonstrated previously that cortisone and chloroquine stabilize lysosomal membranes in vitro [20]. Therefore, pretreatment of cells with such “stabilizers” should protect against “labilization” of lysosomal membranes while inhi14 - 701802

biting enzyme release and subsequent chromosome damage. Additionally, vitamin A, a known labilizer of isolated lysosomal membranes in vitro, was tested for its capacity to cause chromosome damage. MATERIALS AND METHODS Cell cultures Leucocyte-rich plasma (90-95 % small lymuhocvtes) was obtained from n&ma1 healthy donors -and lymphocyte cultures (1 x lOa cells/ml) were initiated in Eagle MEM (Spinner) medium containing 25 % fetal calf serum, penicillin (100 units/ml), streptomycin (100 mg/ml) and 0.1 % pg/ml phytohemagglutinin (PHA, Burroughs-Wellcome, Inc.). Two replicate cultures were incubated for each treatment in all cases, and the results are presented as their mean. Cultures were incubated at 37°C for 72 h, the last 2 h in the presence of 0.05 pg/ml Colcemid (CIBA Pharmaceutical Inc., Summit, N.J.) to accumulate cells in metaphase. At various times during this incubation period, exogenous agents were added to the cultures. Harvest of the cells and slide preparations were achieved by a slight modification of the method of Moorhead et al. [14]. Exptl Cell Res 64

210

M. M. Cohen & Rochelle Hirschhorn

Added exogenous agents

Chromosomal scoring

Due to the insolubility of various lysosomal stabilizers and the vitamin A compounds, Dimethylsulfoxide (DMSO) was used as a solvent (J. T. Baker Chemical Co., Phillipsburgh, N.J., Lot No. 32079). The various drugs were initially dissolved in DMSO with subsequent dilutions in distilled water. The final concentration of DMSO in the cultures did not exceed 1 %. Chloroquine dihydrochloride (CL) (Winthrop Labs, New York, Lot No. ROlSUK) and Cortisone acetate (CA) (Intra Products, Dayton, Ohio, Lot No. VI1 BO l-7479) were added to the lymphocyte cultures at the final concentration of 1 x 10m5M. Vitamin A alcohol (Lot No. 9K-16) and vitamin A acid (Lot No. 7R-1531A. Hoffman LaRoche, Nutley. N.J.) were used as lysosomal membrane labilizers: Final concentrations of the vitamin A alcohol and acid in the lymphocyte cultures were 1O-4, 10-5, and 10-r M, added for 48, 24, and 4 h prior to harvest of the cells. Streptonigrin (SN) (Chas. Pfizer Inc., Brooklyn, N.Y) was dissolved in acetone (1 mgiml) and further dilutions were made with distilled water.. Previous experiments [4, 51 demonstrated that a concentration of 0.01 pg SN/ml for the final 24 h of culture yielded significant chromosomal damage and yet allowed recovery of sufficient analyzable metaphase cells. Mitomycin C (MC) (Bristol Labs, Syracuse, N. Y., Lot No. 58F-606) was dissolved in histilled water and used in the final concentration of 1.0 pg/ml for the last 24 h of culture, as determined by previous experimentation [6]. Experiments using the chromosome breaking agents MC and SN in combination with the lysosomal stabilizers CA and CL were performed as follows: (1) Twenty-four hours prior to harvest of the cells, the chromosome breaking agent (either SN-0.01 pg/ ml or MC-l.0 pg/ml) was added to the cultures followed 6 h later by the lysosomal stabilizer, either CA (1 x 1O-5M) or CL (1 x 1O-5 M). (2) The reverse of the above sequence, e.g. at 24 h prior to harvest, the stabilizer was added followed by the chromosome breaker at 18 h prior to harvest. (3) Both the stabilizer and the chromosome breaking agent were added simultaneously at 24 h prior to harvest.

After harvest, several slides per replicate culture were prepared, stained with 2 % acetorcein, and coded by individuals who did not participate in the microscopic scoring of the cells. It was hoped that a total of at least 100 metaphase cells per treatment would be assessed for chromosomal damage. However, due to the cytotoxicity of some of the agents employed, this number of cells was not always obtained. Well spread metaphase cells were selected under low magnification ( x 250) and chromosomes were scored under oil immersion phase contrast microscopy (approx. x 1 560). Once a cell was selected under low power, it was included in the study. Chromosomal abnormalities were scored as breaks only if an obvious discontinuity of the chromatin was visible. Breaks were classified as chromatid if only one chromatid was affected and as “isochromatid” if both chromatids were broken at the same location. Both of these types of abnormality were scored as single breaks. Singe fragments were included with chromatid breaks, while “double” fragments were scored as isochromatid breaks. Dicentric chromosomes and quadriradial configurations (QR) were considered as containing two breaks. Attenuated, nonstaining chromosomal regions, other than the normal and obvious secondary constriction regions were scored separately as “gaps” but were not included in the calculation of the breakage rates. The data are reported as the mean number of breaks per cell. The rate of cell division (Mitotic Index) was quantitated by counting the number of metaphase cells in 250 leucocytes observed on each of eight slides per treatment. The rate of mitosis therefore, was based on the total of 2 000 cells per treatment.

Physical treatments Cultures were also subjected to variation in temperature. A series of preliminary experiments, varying temperature and length of exposure, were performed to determine optimum conditions for yieldina analvsable cells. Exnosure of lvmahocvte cultures to-temperatures of 60”; 56” and 50°C fo; periods of time ranging from 0.5-5 min all proved highly lethal and yield virtually no mitoses. However, 45°C for 1.5 min, although lethal to some cells, allowed recovery of metaphase cells of sufficient quality and number for analysis. A cold shock of 4°C for 2.5 h was also employed for other cultures. Lymphocytes were treated with 1.Opg/ml MC 24 h prior to harvest. Ten hours later (14 h prior to harvest) the cells were subjected to either the heat or cold treatments. These experiments were repeated three times (each with three replicates). Exptl Cell Res 64

Lysosomal enzyme assay Approx. 5 g of liver obtained from male hybrid albino rabbits were minced in ice-cold 0.25 M sucrose, the pieces were further washed three times in 0.25 M sucrose and homogenized in 50 ml of 0.25 M sucrose in a glass homogenizer with a motor-driven Teflon pestle (Tri-R Instruments, Jamaica, N. Y.) at maximum speed, using six up and down strokes of the pestle. The homogenate was centrifuged at 2 500 rpm in a Sorvall Model SS-3 centrifuge for 10 min. The resulting supernatant was centrifuged at 12 500 rpm for 20 min and the pellet washed by resuspending gently in 25 ml of 0.25 M sucrose and recentrifuged at 12 500 rpm for an additional 20 min. The pellet was then gently resuspended in 50 ml of 0.25 M sucrose and consisted of a “large granule” fraction enriched in lysosomes. Aliquots (2 ml) of this granular fraction were incubated for 1 h at 37°C in the presence of varying concentrations of mitomycin C (5.0-0.1 pg/ml), streptonigrin (0.05-0.001 pg/ml), diluent (water) or 0.1 % Triton-X-100 (Rohm & Haas, Philadelphia, Pa). These were then centrifuged at 20 000 g for 20 min at 4°C and the activity of enzymes released into the suuernatant determined. &Glu&ronidase was assayed by the method of Talalay et al. [18] and acid phosphatase as previously described [9]. The results are expressed as the per-

Lysosomes and chromosome breakage

211

1. Chromosomal damage induced by 0.01 ,uglmE streptonigrin (SN) in combination with the 1 x 10-5M of the lysosomal stabilizers, cortisone (CA) or chlorquine (CL)

Table

Treatmenta

Breaks

Gaps

Number of quadriradials

Total cells

Mean breaks/cell

Control SN CA+SN SN+CA CA SIN

0.040 0.592 0.320 0.224

0.087 0.320 0.160 0.120

0 1 lb 2

300 125 125 125

0.040 0.608 0.336 0.256

0.192

0.144

0

125

0.192

CLlSN SN+CL CL SIN

0.344 0.208

0.232 0.176

0 0

125 125

0.344 0.208

0.192

0.208

0

125

0.192

a First drug added 24 h nrior to harvest and second drug added 18 h prior to harvest. b Represenys a dicentric chromosome rather than QR. centage of enzyme activity released by incubation with Triton-X-100. Human peripheral lymphocyte cultures were established as previously described [3] and stimulated with phytohemagglutinin (PHA) followed by incubation at 37°C for 57 h. At this point, 4 h prior to the harvest of the cells, vitamin A alcohol was added (final concentration 9 x 10e4 M). After a total of 61 h incubation, the cells were separated and fractionated into subcellular fractions by methods previously described [3]. The release of lysosomal p-glucuronidase was measured as above [9] and expressed as percent enzyme released by incubation with 0.1 % Triton-X-100.

RESULTS Effect of cortisone and chloroquine upon streptonigrin and mitomycin C induced chromosome aberrations If chemical agents were to produce chromosome breaks by rupture of lysosomes, the addition of lysosomal stabilizing agents prior to or concomitant with such treatment should protect the chromosomes from damage. Previous studies [7] have indicated that the lysosomal stabilizers themselves caused a marked suppression in mitosis, apparently dose and time related, but no observable increase in chromosome damage. DMSO has no effect, either on the mitotic rate or chromosomal damage, when compared with controls.

Streptonigrin (SN) Table 1 illustrates the effect on chromosome damage induced by SN in combination with the lysosomal stabilizers CA and CL. Addition of 0.01 ,ug SN/ml resulted in a 15-fold increase in chromosomal damage when compared with controls (0.608 breaks/cell vs 0.040 breaks/cell). Fig. 1 illustrates SNinduced chromosomal damage. The presence of either lysosomal stabilizing agents (CA or CL) for the 6 h prior to the addition of SN resulted in frequencies of breaks significantly greater than those found in the controls (0.336 and 0.344) breaks/cell, respectively versus 0.040 breaks/cell). However, a decrease in breaks was observed as compared with treatment with SN alone. An even greater decrease in chromosome damage was achieved by adding the stabilizer 6 h after addition of SN (0.256 breaks/cell for CA and 0.280 breaks/cell for CL) and the most “protective” treatment was observed when either CA or CL was added simultaneously with the SN at 24 h prior to harvest (0.192 breaks/cell in both cases). Thus, although a decrease in chromosomal damage was obtained with the addition of the lysosomal ExptI Ccl1 Res 64

212

M. M. Cohen & Rochelle Hirschhorn

Fig. 1. Chromosomal aberrations induced by streptonigrin (SN): (a) chromatid breaks; @) isochromatid breaks resulting in acentric fragments; (c) dicentric chromosomes; (d) cells exhibiting multiple abnormalities.

stabilizers, it apparently mattered little whether the drugs were added before, after, or at the same time with the SN. Mitomycin

C (MC)

The effect of CA and CL upon MC-induced chromosome damage was similar to that Exptl Cd Res 64

seen with SN (table 2). The control rate of chromosome damage was 0.020 breaks/cell as compared with 2.924 breaks/cell with 1.0 pug MC/ml. As with SN, a reduction in chromosome breakage was observed when MC was combined with the lysosomal stabilizers CA and CL. Also, similar to the SN

Lysosomes and chromosome breakage

213

Table 2. Chromosome damage induced by 1.0 ,uglml mitomycin C (MC) in combination with 1 x 10-5M of the lysosomal stabilizers, cortisone acetate (CA) and chloroquine (CL) Treatment=

Breaks

Gaps

Number of quadriradials

Total cells

Mean breaks/cell

Control MC CA+MC MC+CA CA -/MC CL+MC MC+CL CL .I. MC

0.020 2.618 0.967 2.136

0.060 0.878 0.400 1.049

0 20 19 8

450 131 110 81

0.020 2.924 1.309 2.333

0.991

0.609

10

110

1.173

1.222 1.413

0.911 0.870

21 13

135 92

1.533 1.696

1.284

1.716

5

102

1.382

a First drug added 24 h prior to harvest; second drug added 18 hours prior to harvest.

experiments, the greatest “protection” was achieved when MC and the stabilizers were simultaneously (CA + MC = 1.173 added breaks/cell and CL + MC = 1.382 breaks/ cell). In addition to chromosomal breaks, MC also induces striking quadriradial configurations (QR) with relatively high frequency [6] (fig. 2). In table 2 (line 2), MC alone produced 20 QRs in 137 cells yielding a frequency of 15.2 % of cells containing this readily discernible cytologic marker. There was no absolute protection by the lysosomal stabilizers against QR formation, since these rearrangements were present in all the treated cultures regardless of the time of addition of CA or CL.

are undamaged or minimally damaged might survive to mitosis. Therefore, two physical treatments affecting mitotic rate but not chromosome damage, heat and cold, were studied. Table 3 presents the pooled data of three experiments indicating that both the heat and cold treatments in the control cells caused a marked suppression in mitosis with no increase in chromosome damage. Mitomycin treatment alone, suppressed mitosis and also induced the expected concomitant increase in chromosome damage. This double treatment, combining MC with either heat or cold, caused a further reduction in mitotic rate. More importantly, the frequency of chromosome damage in these cultures was reduced to almost half that observed with MC alone.

Physical treatments Although the addition of CA and CL appeared to diminish the incidence of chromosome breaks, the least “protective” regimen was that which was expected to be most protective, i.e. the pretreatment of cells with the stabilizer. If, however, mitotic suppression were to selectively affect cells with chromosome breakage, only those cells which

Effect of MC and SN upon isolated lysosomes The above results suggest that the lysosomal stabilizers, CA and CL, did not directly protect against chromosome damage, indicating that the particular chromosomebreaking agents under investigation (MC and SN) do not act via the destruction of lysosomal membranes and subsequent enExptl Cell Res 64

214

M. M, Cohen & Rochelle Hirschhorn

Fig. 2. Chromatid exchanges induced by mitomycin C (MC). Both quadriradial (QR) and triradial (TR) configurations are depicted. Most formations are single two chromosome exchanges, but several are complex involving more than two chromosomes.

zyme release. Incubation of lysosomes varying concentrations of MC and SN, ficient to cause chromosome damage, not cause any greater release of either phosphotase or /3-glucuronidase from

with sufdid acid the

lysosomes than did the diluent alone (table 4). Therefore, it would indeed appear that neither chromosome-breaking agent could be demonstrated to have an effect on the integrity of the lysosomal membrane.

Table 3. Effects of combinatory treatments of 1 ,uglml MC with heat (45°C) and cold (4°C)

on mitosis and chromosomal damage Treatment

No. cells

No. breaks

No. QR

No. gaps

Breaks per cell

Percent mitosis

Control Control (4°C) Control (45°C) MC MC+4”C MC+45”C

643 268 300 240 270 339

21 7 6 422 297 401

0 0 0 59 3’;

50 25 29 166 126 196

0.033 0.026 0.020 2.250 1.248 1.372

5.01 3.72 3.19 1.90 0.40 0.52

Exptl Cell Res 64

Lysosomes and chromosome breakage

215

Table 4. Effect of mitomycin C (MC) and streptonigrin (SN) on the release of the lysosomal enzymes /3-glucuronidase and acid phosphatase from rabbit liver lysosomes in vitro Values expressed as percentage of enzyme activity released by 0.1% Triton-X-lOOa Mitomycin C

Streptonigrin

Concentration 0-a MC/ml)

Beta glucuronidase

Acid phosphatase

Concentration (.m SN/mU

5.0 2.5 1.0 0.5 0.1 Water

8.0 8.6 8.9 8.7 8.7 9.2

5.7 5.3 5.3 5.0 5.2 6.0

0.050 0.025 0.010 0.005 0.001 Water

Beta glucuronidase 9.6 9.9 9.2 10.3 9.2 9.5

Acid phosphatase 5.5 3.9 5.0 5.5 5.8 5.4

a Average value of @-glucuronidase released by 0.1 % Triton-X-100 = 0.632 PM/ml/h. Average value of acid phosphatase released by 0.1 % Triton-X-100 = 3.09 PM/ml/h.

Vitamin A compounds Vitamin A compounds are known to rupture isolated rabbit lysosomes [20]. Preliminary experiments suggest that the addition of vitamin A alcohol to intact human lymphocytes also increases the permeability of the lysosomal membrane so that more enzyme activity is found in the supernatant fraction (table 5). However, no chromosome-breaking potential for the three concentrations of either vitamin A alcohol or vitamin A acid (lop4 M; 1O-5 M; and lo-’ M) for 48, 24, and 4 h exposures, was demonstrated when compared with controls. Additionally, in contrast to the findings with most other

exogenous agents, these vitamin A compounds apparently did not cause a suppression of mitosis (fig. 3). DISCUSSION Several agents capable of causing chromosomal aberrations, such as ultraviolet and

14

I

l--l

6

Table 5. Effect of vitamin A alcohol (9 x IOm4M) upon the subcellular distribution of /3glucuronidase activity in cultured human lymphocytes

r

12

Control /

Cell fraction

10

Percent of total enzyme activity Vitamin A alcohol

Control

8

‘4

---/ a‘-

,*--

’ ,*--

.I0M -4

b Mean

Debris 10.4; 9.6 Nuclear and 51.2; 51.3 Granular Supernatant 32.4; 39.0

Mean

10.0

6.5; 10.5

8.5

54.4

63.0; 66.1

64.5

35.7

30.4; 21.4

25.9

6 t

I 4

I 24

I 48

hours prior to harvest; ordinate: % mitosis. The effect of (a) vitamin A alcohol, and (6) vitamin A acid on the mitotic rate of human lymphocyte cultures. Fig. 3. Abscissa:

ExptI Cell Res 64

216 M. M. Cohen & Rochelle Hirschhorn X-irradiation as well as some viruses, also cause the release of lysosomal enzymes by membrane disruption. Allison & Paton [l] attempted to selectively rupture lysosomes by the addition of vital dyes such as acridine orange and neutral red, which are concentrated in lysosomes. Upon photoactivation of these concentrated dyes, the lysosomal membrane was preferentially damaged. An increase in chromatid breaks was found following such treatment, and it was therefore suggested that chromosome damage may be induced by the release of lysosomal enzymes. The possible involvement of this mechanism in virally-induced chromosomal damage was investigated by Aula & Nichols [2]. Both measles virus and the human adenovirus 12 were studied in human leucocytes as well as in heteroploid tissue culture lines. An attempt was made to protect against chromosome damage by the addition of a lysosomal stabilizer (cortisone acetate), but in no case was protection evident. Additional experiments, using adenovirus 12 in five different cell lines of human, rodent and marsupial origin, suggested that chromosomal damage inflicted by the virus is probably under the control of the viral genome and is unlikely to involve the activation of cellular lysosomal enzymes [21]. Therefore, the involvement of lysosomal rupture and enzyme release was not suggested as an integral part of the mechanism of chromosome damage as induced by these viruses. The present investigations have attempted to evaluate the possible role of lysosomes in chemically-induced chromosome breakage. Previous work would suggestthat mitomycin C, when injected into sarcoma-bearing animals, causes a labilization of lysosomes in the sarcoma cells [15]. However, the possibility of intraperitoneal admixtures with cell types other than sarcoma cells must be considered as an alternative explanation for the Exptl Cell Res 64

apparent change in lysosomal enzyme-distribution. The results of direct enzyme assays on isolated rabbit lysosomes (table 3) indicate that MC does not directly cause lysosomal membranes to rupture in this particular system. Although no direct evidence for lysosomal damage by either SN and MC was obtained, there was an apparent decrease in chromosomal damage when these drugs were combined with the lysosomal stabilizers (tables 1, 2). The greatest decrease in damage was achieved by adding the stabilizer and the chromosomal “breaker” simultaneously. Addition of the stabilizer 6 h after MC also reduced cytogenetic damage. The least “protective” regimen was that which was expected to be most protective, i.e. the pretreatment of cells with the stabilizer. Thus, although it is tempting to infer that prevention of lysosomal labilization by the addition of CA and CL did indeed prevent chromosomal aberrations, several other explanations were examined. Both MC and SN have previously been shown to markedly suppress mitosis [4, 61. Previous studies indicate a similar mitotic inhibition for the lysosomal stabilizers tested [7]. If those cells which are chromosomally damaged by MC or SN are still further inhibited to divide by the presence of lysosomal stabilizers, it may well be that only those cells which are relatively undamaged or slightly damaged actually survive to mitosis. This hypothesis was tested by utilizing two arbitrary treatments which were expected to reduce the mitotic rate (namely, heat and cold), but which would not be expected to stabilize lysosomal membranes. These treatments also reduced the observed frequency of MC induced chromosome breaks (table 3). These data suggest, therefore, that selection against cells with greater chromosome damage occurs; resulting in (1) a reduction in mitotic rate and (2) an apparent reduction of chromosome dam-

Lysosomes and chromosome breakage age secondary to preferential survival of “healthier” cells. Therefore, this would in effect reduce obvious chromosomal damage in those mitotic cells observed after the combination treatments regardless of the sequence in which the drugs are added. Additionally, other factors may play a role in the reduction of chromosome damage seen. Competitive inhibition may exist, since it has been suggested that both SN and MC interact with DNA [ll, 171. Similar observations have been made with chloroquine [8, 161. If such be the case, and both the “breakers” and “stabilizers” compete for the same active sites, overall chromosomal damage may be reduced by virtue of the fact that the lysosomal stabilizers alone do not break chromosomes [7]. Yet another explanation lies in the possibility that a direct interaction may occur between the chromosome-breaking agent and the lysosomal stabilizer rendering the MC or the SN inactive, thereby lowering the overall frequency of chromosomal damage. Even though these suggestions have not been proven, other explanations besides the stabilization of lysosomal membranes may readily explain the decrease in chromosome damage upon addition of the stabilizer. On the other hand, the investigations with vitamin A alcohol and vitamin A acid, known lysosomal labilizers, failed to yield evidence in support of the hypothesis. Enzymatic assays in various systems (table 5) and an electron microscopic study [7] demonstrated that the lysosomes were indeed ruptured following vitamin A treatment. However, there was no suggestion of increased chromosomal damage induced by vitamin A compounds. If the original hypothesis [l] is valid, vitamin A, as well as other lysosomal labilizers, should cause chromosomal breaks.

217

The technical assistance of Mrs Elizabeth Kovi and Mrs Clara Lockwood is greatly appreciated. We are also indebted to Dr Kurt Hirschhorn for his helpful discussions during the course of this study and critical reading of the manuscript. This investigation was supported in part by grants from the US Children’s Bureau (Projedct No. 417) and the New York Heart Association. R. H. is a Senior Investigator of the New York Heart Association.

REFERENCES 1. Allison, A C & Patton, G R, Nature 207 (1965) 1170. 2. Aula. P & Nichols. W W. Exotl cell res 51 (1968) 595. ’ ’ 3. Brittinaer. G. Hirschhorn. R. Douglas. S D & Weissmad, G; J cell biol 37 (1968) 4i2. ’ 4. Cohen, M M, Shaw, M W & Craig, A P, Proc natl acad sci US 50 (1963) 16. 5. Cohen. M M. Cvtosenetics 2 (1963) 271. 6. Cohen; M M’& Shaw, M W, J cellbiol 23 (1963) 386. 7. Cohen, M M, Hirschhorn, R & Freeman, A F, Genetic concepts and neonlasia. Williams & Wilkins, Baltimore (1970). 8. Cohen, W N & Yielding, K L, Proc natl acad sci US 54 (1965) 521. 9. Hirschhorn, R, Hirschhorn, K & Weissman, G, Blood 30 (1967) 84. 10. Iyer, V N & Szybalski, W, Proc natl acad sci US 50 (1963) 355. 11. Kersten, H, Biochim biophys acta 55 (1962) 558. 12. Kersten, H, Kersten, W, Leopold, G & Schieders, B, Biochim biophys acta 80 (1964) 521. 13. MacGregor, H C & Callan, H G, Quart j microstop sci 103 (1962) 172. 14. Moorhead, P S, Nowell, P C, Mellman, W J, Battius. D M & Hungerford. D A. Exutl cell res 20 (1966) 613. . 15. Nitani, H, Suzuki, A, Shimoyama, M & Kimura, K, Gann 57 (1966) 193. 16. Parker, F S & Irvin, J L, J biol them 199 (1952) 889. 17. Radding, C M, Genetics today, p. 22. Pergamon Press, Oxford (1963). 18. Talalay, P, Fishman, W H & Huggins, C, J biol them 166 (1946) 757. 19. Venaut, A M, Theron, M C, Bresson, M L & Cattan, A, Rev franc clin biol 13 (1968) 707. 20. Weissman, G, The interaction of drugs and subcellular components of animal cells, p. 203. Churchill, London (1968). 21. Zur Hausen, H, J virol 2 (1968) 218.

ReceivedJune 18 1970 9

Exptl Cell Res 64