10T12 clone 8 cells

Mutation Research, 170 (1986) 133-143

133

Elsevier MTR 01064

Morphological transformation and chromosome damage by amsacrine in C 3 H / 1 0 T 1 clone 8 cells L y n n e t t e R. F e r g u s o n *, P i e r r e v a n Zijl a n d S t e p h e n N e s n o w

a,**

Cancer Research Laboratory, University of Auckland School of Medicine, Private Bag, Auckland (New Zealand) and a Carcinogenesis and Metabolism Branch, Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (U.S.A.

(Received 1 October 1985) (Revisionreceived16 January 1986) (Accepted24 January 1986)

Summary Morphological transformation, cell survival, chromosomal aberrations and micronuclei were measured in C3H/10½CL8 cells after 24 h exposure to amsacrine. A weak but dose-related increase in the percentage of dishes containing transformed foci occurred. As previously reported for alkylating agents, this effect was increased by treating 5 days instead of 1 day after plating. There was no evidence for gene mutation at the N a / K ATPase locus, although amsacrine induced micronuclei in a large percentage of cells and chromosomal aberrations, including interchange events and double minute chromosomes, in dividing cells. In would appear that transformation and chromosomal events may be related in amsacrinetreated C3H/10T½CL8 cells. The results strongly suggest that amsacrine has carcinogenic potential, possibly related to its chromosome-breaking properties.

Amsacrine (4'-(9-acridinylamino)methanesulfon-m-anisidide is a 9-anilinoacridine derivative, developed as an antitumour agent by the late Dr. B.F. Cain (Cain and Atwell, 1974). It is currently used in the clinic for the treatment of leukaemia (Omura et al., 1983) and lymphoma (Cabanillas et al., 1981) and is gaining increasing acceptance as initial therapy for the first of these indications. Many of the current cancer treatments can themselves cause cancer, and this is becoming an increasing problem for long-term survivors of both radiotherapy and chemotherapy (Harris, 1976; IARC, 1981). For a relatively new agent such as amsacrine, it is clearly of value to know whether it * To whom requests for reprints should be addressed. ** The research described in this paper does not necessarily reflect EPA poli9y.

will or will not be a carcinogen, and how this potential rates in relation to other clinical agents. Amsacrine is a DNA-binding agent, intercalating into DNA but not chemically reacting with it (Baguley et al., 1981). It induces frameshift mutations in S a l m o n e l l a t y p h i m u r i u m strains TA1537 and TA97 among others, but not in TA98 (Ferguson and Denny, 1979; Ferguson et al., 1985). In Chinese hamster V79 cells, it induces mutations to 6-thioguanine, but not ouabain resistance (Wilson et al., 1984). It stimulates sister-chromatid exchange (Crossen, 1979) and has been demonstrated as a clastogen in several different mammalian cell lines (Deaven et al., 1978; Ferguson and Baguley, 1984). On the basis of these data, it might be predicted that amsacrine possesses carcinogenic potential. Although a bioassay for development of adenomas in strain A mouse lung

0165-1218/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)

134 after amsacrine treatment gave negative results (de la Inglesa et al., 1984), a negative result in this model does not provide unequivocal evidence of non-carcinogenicity (Maronpot et al., 1983). Many antitumour agents induce morphological transformation in C3H/10T½ CL8 ( C 3 H / 1 0 T ½) cells, and this correlates closely with their carcinogenic activity (Benedict et al., 1977; IARC, 1982, Heidelberger et al., 1983). For many chemicals, treatment of C3H/10T½ cells 5 days after seeding produces an enhanced transformation response (Nesnow et al., 1983, 1985). We have therefore used both the standard and 5-day protocol to investigate morphological transformation by amsacrine and correlated these data with the production of chromosome damage measured both as aberrations per se and as micronuclei, as well as with induction of gene mutation, measured as resistance to ouabain. Materials and methods

Chemicals Benzo[a]pyrene was purchased from Aldrich Chemical Co., Milwaukee, WI (U.S.A.). The isothionate salt of amsacrine was kindly provided by Dr. B.C. Baguley, Auckland Division Cancer Society of New Zealand. Fresh solutions of both compounds, amsacrine dissolved in 50% ethanol and benzo[a]pyrene in acetone, were prepared immediately before use. Cells and culture conditions The mouse embryo fibroblast line C3H/10T½ CL8, derived by Reznikoff et al. (1973), was used in these experiments. Cell cultures were maintained in humidified incubators with an atmosphere of 5% CO 2 in air at 37°C. All cultures were grown in Eagle's basal medium with Earle's salts and L-glutamine supplemented with 10% heat-inactivated foetal bovine serum (Gibco New Zealand Ltd.). The line was free of Mycoplasma throughout these experiments, as judged by cytochemical staining (Chen, 1977). No antibiotics were used in the medium throughout the experiments. Cell-transformation assay Seeding densities were 2000/dish (or higher to compensate for toxicity) if dishes were exposed to

drug after I day, or at 1000/dish for dishes treated on day 5. Cells were seeded onto 60-mm Petri dishes (Corning, Coming, NY) in 5 ml of medium (24 dishes per experimental point). After the time specified, they were treated with chemical in 20/al of solvent or with 50% ethanol as the negative control for 24 h as described by Reznikoff et al. (1973) and Nesnow et al. (1983). After 24 h treatment, the medium was removed and replaced with fresh medium, again containing 10% foetal bovine serum without antibiotics. This medium was changed weekly until the cells reached confluence. The fetal bovine serum concentration was reduced to 5%, and weekly medium changes were continued. At the end of 6 weeks, the dishes were washed with 0.85% NaC1 solution, fixed with methanol, stained with Giemsa, and scored for morphological transformation. All 3 different types of foci, as previously described for C3H/10T½ cells following treatment with polycyclic aromatic hydrocarbons, were scored. These data are presented as the numbers of type I, type II and type III foci per number of dishes scored and also as the percentage of dishes that exhibited type II or type III loci. (These latter two types are considered more important than type 1, as 50-80% of these loci produce sarcomas when injected into irradiated C3H mice, Reznikoff et al., 1973) Survival assays Cytotoxicity assays were run concurrently with the transformation assays using two different methods. Method 1 used the same protocol as the transformation assays, except that the dishes were seeded with 200 cells, there were 5 dishes per experimental point, and the dishes were stained 10-12 days after plating. This permitted an estimate of the number of clones which survived drug treatment. In Method 2, 2 x 103 cells (or higher to compensate for toxicity) per 60-mm dish were plated (1 x 103 for 5-day exposure), and, after drug exposure the cells were trypsinised, counted using a haemocytometer and seeded in fresh dishes at 200 counted cells per dish. Again there were 5 dishes per experimental point and the dishes were stained 10-12 days after plating. This modified methodology permitted an estimate of number of cells surviving drug treatment.

135

Statistical analysis of transformation data The raw data were fitted to a multiple logistic model to test for dose effect (Nesnow et al., 1981). In these analyses, the probability of a dish having a least one type II or type III focus ( P ) is taken to be

tonic treatment with 0.075 M KC1. The chromosomes were stained with Giemsa, and 50 metaphases for each concentration tested were analyzed for different chromosomal aberrations, including chromatid breaks, gaps, segments, chromatid exchanges and dicentric chromosomes (UKEMS, 1982).

p= 1 + e x p ( - ( f l I + fl2(dose)+ fl3(dose)2)) Using maximum likelihood methods, t h e parameters ill, r2 and f13, as well as the asymptotic variance-covariance matrix, were estimated. A X2 goodness-of-fit statistic was used to evaluate the model's ability to fit the data, and a likelihood ratio test was used to assess the strength of the dose effect.

Mutagenesis assay This procedure followed that of Evans et al. (1981). Amsacrine-treated and control cells were plated at various densities in 15 x 100 mm petri dishes. When the cells had attached to the plastic surface, the growth medium was replaced with medium containing 1 mM ouabain (Calbiochem). The plates were then incubated for 2 weeks at 37°C, after which time the cells were stained and the ouabain-resistant colonies counted.

Chromosomal studies The cells were plated with the same cell densities and conditions as for transformation assays. 1 day or 5 days later, the compounds were added for 24 h, the cells washed with PBS and fresh warmed medium added. For micronucleus studies, the cells were harvested a range of times after this medium change in preliminary experiments, or a single time (24 h after) in subsequent work. Cells were prepared according to methods previously described (Wilson et al., 1984). Trypsinized single-cell suspensions were fi~ed in ice-cold Carnoy's fixative (methanol:acetic acid, 3:1, v / v ) after swelling in hypotonic KC1 (0.075 M) for 10 min at 37°C, and were dropped 20 cm onto clean glass slides. Cytoplasmic structures were scored as micronuclei if they showed the same Giemsa-staining reaction as the nucleus, were clearly resolved from the nucleus (to distinguish from nuclear blebs), and had diameters in the range 2.5-10/zm. The range of nuclear diameters in these slides was 20-44/~m for control cells. Either 100 cells with micronuclei or 2000 cells in total were scored for each data point. For chromosomal analyses, colchicine (0.4 /zg/ml) was added 2 h before harvest. Chromosome preparations were made according to standard techniques (UKEMS, 1982). Briefly, the cells were fixed with 3 changes of ice-cold methanol:acetic acid (3:1) after a 15-min hypo-

Results

Cell transformation The ability of amsacrine to induce morphological transformation in C3H/10T½ CL 8 cells is summarised in Table 1. Data for benzo[a]pyrene as positive control are also given in Table 1. There was a clear, dose-related increase in transformed colonies after amsacrine treatment, and this effect was enhanced by treatment at day 5 rather than day 1. Compared with benzo[a]pyrene, amsacrine causes more type I and II colonies in relation to type III colonies. Nonetheless, amsacrine does reproducably cause the type III foci, previously associated with sarcoma formation in irradiated mice (Reznikoff et al., 1973). Statistical analysis indicated that both the 1-day and 5-day studies showed statistically significant dose effects. The significance probability associated with the likelihood ratio test was 0.02 for the 1-day study and < 0.001 for the 5-day study.

Mutagenesis Benzo[a]pyrene as positive control increased the frequency of ouabain-resistant colonies in C3H/10T½ cells, to 2.3 x 10 -s (day-1 treatment) and 2.5 x 10 -s (day-5 treatment). Values for the negative control and for amsacrine-treated cells were all less than 1.5 × 10 -6.

136

TABLE 1 MORPHOLOGICAL TRANSFORMATION IN C3H/10T~2 CELLS AFTER EXPOSURE TO AMSACRINE FOR 24 h Transformation Total number of loci/Number of dishes scored

% of dishes with type II and type III foci

Type I

Type II

Type llI

Negative control (ethanol, 2 #g/ml)

4/54

0/54

0/54

0%

Positive control (benzo[ a ]pyrene, 1 ~g/ml)

48/43

43/43

17/43

69.7

0/16

0/16

0/16

0

5

2/18

1/18

0/18

5.6

10

5/57

3/57

0/57

5.6

20

49/67

8/67

1/67

7.5

40

50/64

5/64

0/64

7.8

80

30/34

5/34

1/34

11.8

160

16/30

8/30

3/30

13.3

Negative control a (ethanol, 2 t~g/ml)

3/66

0/66

0/66

0

Positive control b (benzo[ a ]pyrene, 1/zg/ml)

57/64

75/64

22/64

78.0

Amsacrine (ng/ml) 10

26/55

2/55

0/55

3.6

20

34/55

5/55

0/55

9.1

40

42/63

18/63

1/63

9.5

Day-1 treatment

Amsacrine (ng/ml) 2.5

5-day treatment

80

51/60

11/60

2/60

18.3

160

48/63

18/63

3/63

23.8

a The plating efficiency in solvent control was 29%. b Survival after treatment with benzo[a]pyrene was 67%.

Survival

C h r o m o s o m e studies

M e a s u r e m e n t s of cell survival (as m e a s u r e d with m e t h o d 2) a n d of the n u m b e r of p l a t e d clones surviving d r u g e x p o s u r e ( m e t h o d 1) are p l o t t e d on Fig. 1. A l t h o u g h sensitivity is a p p r o x i m a t e l y equal w h e t h e r cells are treated I d a y or 5 d a y s after plating, as might be expected, clonal survival ( m e t h o d 1) is c o n s i d e r a b l y higher with the latter t r e a t m e n t time.

T h e effects of varying time of harvesting cells for micronuclei was tested after 24 h exposure to 3 different c o n c e n t r a t i o n s of a m s a c r i n e for day-1 a n d day-5 e x p o s u r e c o n d i t i o n s (Fig. 2). These d a t a show that micronucleus i n d u c t i o n reaches a maxim u m a p p r o x i m a t e l y 2 d a y s after d r u g t r e a t m e n t has c o m m e n c e d for low doses, 3 d a y s at higher doses, in b o t h day-1 or day-5 treated cells. D a y 2

137 w a s c h o s e n as a c o n v e n i e n t t i m e f o r m i c r o n u c l e u s m e a s u r e m e n t in s u b s e q u e n t e x p e r i m e n t s .

c o n c u r r e n t l y with the t r a n s f o r m a t i o n dishes for two experiments. Examples of the types of micro-

Micronuclei were estimated from plates treated

nuclei f o r m e d are illustrated in Plates 1 - 3 . C h r o -

TABLE 2 CHROMOSOMAL ABERRATIONS IN C3H/10T½ CELLS PRODUCED BY EXPOSURE TO AMSACRINE FOR 24 h Chromosome changes a Total number of cells with aberrations c 1-day treatment Negative control (ethanol, 2 #g/ml)

Mitotic b Number of cells with chromosome or chromatid exchange

Number of cells with multiple lesions d

index (%)

0

0

0

4.16

23

10

7

1.77

9

0

0

3.91

5

13

2

0

4.02

10

21

11

1

3.23

20

22

12

7

2.55

40

29

12

9

1.92

80

43

4

31

0.95

160

46

6

38

0.87

0

0

0

4.80

22

5

12

1.43

6

0

0

4.43

5

7

0

0

4.39

10

13

2

2

3.54

20

24

3

4

2.42

40

34

7

20

2.41

80

46

3

35

1.93

160

48

2

43

1.21

Positive control (benzo[ a ]pyrene, 1/~ g/ml) Amsacrine (ng/ml) 2.5

5-day treatment Negative control (ethanol, 2/zg/ml)

Positive control (benzo[ a ]pyrene, 1/t g/ml) Amsacrine (ng/ml) 2.5

a b c d

Events per 50 cells. Tetraploid cells have been excluded from analysis. The figure given represents the number of metaphase cells per 1000 cells scored. Numbers represent an average over 3 samples. Gaps have been excluded from this score. These include interchange events and premature chromosome condensation. A cell is scored as showing multiple events when there are too many to be able to distinguish individual lesions.

138 100

100

8o

80

6o

60

:::) 40

40

._J

< _>

>

n'-

O0 20

20 e

t

10

100

200

I

I

i

10

100

200

CONCENTRATION (ng/ml) Fig. 1. Cell (Method 2, O) and colony (Method 1, O) survival (as defined in the 'materials and methods') after treatment with amsacrine 1 day (panel A) or 5 days (panel B) after plating. The data represent an average of 5 plates, from a single experiment.

mosomal changes were also measured in one experiment, in order to provide some correlation between direct measurement of chromosomal change and micronucleus assays. The chromosome data are summarised in Table 2, and total micronucleus formation for day-1 as compared with day-5 treatment shown as a function of drug dose on Fig. 3. 80

At doses of amsacrine at which no cell transformation was detected (2.5 n g / m l or less) there were also no multiple micronuclei formed, no measureable chromosomal interchanges, nor any metaphase spreads showing multiple events. At all other doses of amsacrine both complex aberrations and multiple micronuclei are formed. Amsacrine frequently

80i

A

B

60 m

iii 0 Z

4O

0 4© nO

a~ 20

2

3

4

5

n

n

n

n

o

1

2

3

4

5

DAYS AFTER DRUG TREATMENT Fig. 2. The effects of increasing time after drug treatment on detection of micronuclei in cells treated with amsacrine either 1 day (panel A) or 5 days (panel B) after plating. Numbers are from 2000 cells, from a single experiment. Panel A: ©, control; zx, 20 ng/ml; o, 40 ng/ml; &, 80 ng/ml. Panel B: O, control; zx, 10 ng/ml; O, 40 ng/ml; A, 160 ng/ml.

~

~iii~

~

~

~

~

~ ~!iiiii~i;~

i~ ~i~ ii

ii!~! i~ii~ i

)lates 1-3. C3H/10T12 cell nuclei after treatment with amsacrine. (1) Lobed nucleus. (2) Lobed nucleus with a single micronucleus. (3) Nucleus with multiple micronuclei.

~

...... ~ iiii!¸ iiii!iliiii iiii i!!i! !i!~!i¸ !i !~i!!iiiiiii .......... i i~ii !i!ii~ ii!i?~ ~ 5 + ~ { ~ 7 ~ ? ~

140

00

,J

'A

//~

Q

•

/

/

~/

80 w (.9 < 60

Z

~

W

•

o: 4O w £i. 20

~

&

•

o

~

Q

60 40 20 0 % SURVIVAL Fig. 4. The relationship between transformation (O), c]astogenesis (as the total percentage of ceils with micronuclei) ([3) or % of aberrant cells (z~) and cell survival after amsacrine treatment 1 day (open symbols) or 5 days (closed symbols) after plating. 80

F

% m

II

B i Plate 4. A chromosome spread from cells treated with amsacrine, showing numbers of double minute chromosomes and ring chromosomes.

A

o

induced double minutes in this cell line (Plate 4) and there were high numbers of cells in which multiple events occurred. There were multiple aberrations and micronuclei in up to 60% of the cells at doses killing more than 60% of the cells. An average value for micronuclei for benzo[a]pyrene-treated cells was 10.8% for day-1 treated and 14.1% for day-5 treated cells. Although complex aberrations were formed by benzo[a]pyrene, there were not the high numbers of double-minute chromosomes generated by this compound. B

60

,o

40

n~

20

2o

o

o

Q

IO

Q

i

,oo 2~o

CONCENTRATION

N

i

,~o 200

(nglml)

Fig. 3. The effects of increasing concentration of amsacrine on micronucleus induction in cells treated either 1 day (panel A) or 5 days (panel B) after plating. Data represent the average and range of two experiments. O, the percentage of cells containing multiple micronuclei; e , The percentage of cells containing single or multiple micronuclei.

141 60

o 2o

o

'

1()

'

2Lo

'/

JTIO

'

8Jo

% CELL TRANSFORMATION Fig. 5. The relationshipbetween transformationand clastogenesis (as the total percentageof cells with micronuclei)in cells treated with amsacrine (O) or benzo[a]pyrene(11), 1 day (open symbols) or 5 days (closed symbols)after plating.

Relationship between cytotoxicity, chromosome damage and cell survival Data for micronucleus formation, chromosomal aberrations and cell transformation by amsacrine are plotted as a function of cell survival on Fig. 4. There is a direct relationship between each of these events. The relationship is different for benzo[a]pyrene-treated cells. For example, the micronucleus data are shown in relation to transformation frequency for these two agents on Fig. 5. Discussion

The most important result seen in these experiments is that the cancer chemotherapeutic agent, amsacrine, is an effective inducer of morphological transformation in C3H/10T½ cells. This transformation assay has been demonstrated to be an excellent predictor of carcinogenesis in vivo, with around 90% of agents giving positive results in cell transformation assays also giving positive results in animal systems (Heidelberger, 1983). The data therefore call into question previous negative data in the lung adenoma bioassay (de la Ingelesa et al., 1984). Maronpot et al. (1983) considered the ability of the mouse A pulmonary tumor bioassay model to predict carcinogenesis in long-term rodent bioassay studies. Of chemicals tested in both models, the strain A mouse test system correctly predicted the carcinogenicity (or lack thereof) for

20 (37%) of the chemicals. Whether it is some artifact of toxicity, or of metabolism which prevents this potential carcinogen from inducing cancers in this animal model, or whether the lung adenoma model is an inappropriate one for amsacrine could only be established by other carcinogenicity bioassays. Evans et al. (1981) studying ethyl methanesulphonate and Peterson et al. (1981) studying benzo[a]pyrene correlated ability to cause cell transformation in C3H/10T½ cells with ability to cause gene mutation (as measured by increase in resistance to ouabain) in the same cells. Both groups showed a correlation between the two sets of data, and suggested that transformation events are related to gene mutation by these compounds. Peterson et al. (1979) performed similar experiments, correlating cell transformation in C 3 H / 10T½ cells with mammalian mutagenesis in V79 cells for a range of alkylating agents. We have previously published data for mutagenesis by amsacrine in V79 Chinese hamster cells in relation to micronucleus data (Wilson et al., 1984). For either acute or chronic exposure conditions, amsacrine is an effective clastogen and causes a low frequency of increased mutation to 6-thioguanine but not ouabain resistance. The correlation between clastogenesis and mutagenesis in these experiments suggested that the 6-thioguanine resistance might have been caused by a chromosomal rearrangement. In the present study, we have performed preliminary experiments measuring ouabain resistance after treatment with amsacrine in C3H/10T½ cells (Peterson et al., 1981). At significantly cytotoxic doses we have been unable to retrieve any ouabain-resistant colonies from amsacrine-treated cells, although benzo[a]pyrene as positive control significantly increased ouabain-resistant colonies above the untreated control value. The spectrum of genetic and related damae such as micronuclei observed for benzo[a]pyrene and other alkylating agents in C3H/10T½ is different from that shown by amsacrine, where chromosomal events are more pronounced. Whether amsacrine induces cell transformation through chromosomal damage or whether chromosomal events and morphological transformation are parallel events cannot be resolved at this time.

142 Comparison of Table 2 and Fig. 3 suggests that direct measurement of c h r o m o s o m e damage or estimates of micronucleus formation give information with the same sensitivity. There are more than 70 chromosomes in this cell line, and it is laborious to find metaphase spreads which can be unamigously interpreted with respect to chrom o s o m e damage. Micronuclei however, require less labour to prepare and interpret. Particularly with this cell line, they provide a more readily interpreted and measured index of c h r o m o s o m e damage than direct estimates of aberrations. In addition, when the low mitotic index of the cell line especially after amsacrine treatment, is considered, rrficronucleus measurements are relevant to a far higher proportion of the cells than are aberrations data. It is of some interest to compare the micronucleus and chromosomal data presented in this paper with previously published results for V79 cells (Wilson et al., 1984). Micronuclei were formed in up to 60-70% of the cells in the present experiments, whereas the m a x i m u m numbers seen in V79 cells was around 15%. It seems that C 3 H / 1 0 T ½ cells can survive at least as far as cell division with higher numbers of chromsomal aberrations than V79. This might be related to the increased c h r o m o s o m e n u m b e r of C 3 H / 1 0 T ½ cells compared to V79 cells. It is also of interest to note that the high numbers of double-minute chromosomes generated by amsacrine in C 3 H / 1 0 T ½ cells have not been observed in V79 cells (L. Ferguson, unpublished observations). Some aspect of their cell-cycle kinetics,, D N A - r e p a i r mechanisms or differences in metabolism could account for these differences in sensitivity between the two cell lines, The genetic consequences of the much higher proportion of multiple micronuclei in C 3 H / 1 0 T ½ cells is as yet unresolved. As previously seen (Nesnow et al., 1983, 1985) the frequency of cell transformation in C 3 H / 1 0 T ½ cells is enhanced by treating at day 5 rather than day 1, although the cells are no more sensitive to either micronucleus or chromosome abberration formation or cytotoxicity at this stage. In comparison with other (although unrelated) clinical antitumour agents such as bleomycin, melphalan, methotrexate or cyclophosphamide (Benedict et al., 1977, 1978), amsacrine is weak in terms of

m a x i m u m percentage of transformed cells formed, although it is dose potent in its effects. Amsacrine is itself now well established as a clinical antitumour agent, a second-generation analogue is under consideration for clinical trial (Baguley et al., 1984), with others proceeding through preclinical toxicology. It will be of value to determine whether any of these have lower potential than amsacrine for transforming cells. F r o m the data presented in this study, treatment at 5 days after plating is the preferred method for detecting transformation by amsacrine, and micronuclei the most efficient method for detecting c h r o m o s o m e damage. These approaches will be used in subsequent studies with this series of antitumour agent.

Acknowledgements We wish to thank Dr. A. Stead for the cal analysis, and Dr. B.C. Baguley for discussions. This project was funded by from Auckland Division, Cancer Society Zealand.

statistihelpful a grant of New

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