A Tissue Culture Cytotoxicity Test for Large-Scale Cancer ... · ToPLI~r--Tissue Culture Test for...

A Tissue Culture Cytotoxicity Test for Large-Scale Cancer Chemotherapy Screening*

IRVING TOPLIN

(John L. Smith Memorial for Cancer Research, Chas. Pfizer & Co., Inc., Maywood, N.J,)

Recent reviews of the role of tissue culture in cancer chemotherapy screening (5, 6) have empha- sized the need for a more extensive trial of cell cultures as a screening tool for the detection of anti-tumor substances.

Eagle and Foley (~--4) have reported the cytotoxic activity against human cell cultures of more than 180 selected compounds. These compounds had previously been tested against at least three experimental animal tumors. I t was found that 79 per cent of the substances active against two or more experimental tumors showed cell culture ac- t ivity at 10 -4 gm/ml or less. This degree of sensi- t ivi ty was achieved at the cost of ~1 per cent of the tumor-negative compounds also exhibiting cell culture activity at the arbitrary cut-off point of 10 --4 gm/ml or less. I t was also observed that there was no regular difference in the susceptibility to the various drugs of the cell lines derived from malignant and normal tissue. Eagle and Foley concluded that a large-scale investigation of the usefulness of human cell cultures as a cancer chemotherapy screening procedure was warranted.

Ni t ta (7-9) has tested 38 antibiotics and syn- thetics of varying anti-tumor activity against HeLa cell cultures. I t was found that, in general, the strong tumor-inhibitory antibiotics were destructive to HeLa cells at the lowest concentrations, while those antibiotics inactive against animal tumors were least injurious to the cell cultures. Ni t ta concluded that the test method with the use of HeLa cells can be practically applied to the screening or assay of the anti-tumor activity of substances.

The large-scale screening program of the Cancer Chemotherapy National Service Center (CCNSC) with experimental animal tumors affords a unique opportunity to test concomitantly the reliability

* These studies were carried out under Contract SA-43-ph- 1926 between the National Cancer Institute and Chas. Pfizer & Co., Inc. Presented in part at the 50th Annual Meeting of tile American Association for Cancer Research, Atlantic City, April 1~, 1959.

Received for publication May ll, 1959.

and sensitivity of a cell culture screen in detecting anti-tumor agents from a large random group of test candidates. This report describes a simple, rapid, and inexpensive tissue culture cytotoxicity test used in this laboratory to supplement the experimental tumors in the screening of fermentation broth filtrates, natural products, and syn- thetics. Results obtained by this technic for a group of compounds of known tumor activity are also presented. Some preliminary observations are made on the applications and limitations of the test in random screening and in following the fractionation and concentration of anti-tumor agents from crude preparations.

MATERIALS AND METHODS

The tissue culture test used in these studies included three essential steps:

1. Dispensing of graded doses of test solution directly into disposable plastic containers by a microburet technic.

~. Addition of a HeLa or other human cell suspension standardized at 10,000-15,000 cells/ml in growth medium to a total volume of 1 ml/con- tainer.

3. Microscopic evaluation of cytotoxicity after 5 days' incubation by a prescribed cytotoxicity rating system.

Details of the procedure were as follows: Preparation of cell suspension.--Stock cultures of the HeLa

and other human cell strains were grown on the glass surface of Blake bottles in Eagle's medium (1) with 10 per cent pooled human serum and 50 units/ml penicillin and 50 ~g/rnl dihydro- streptomycin. Stock cultures were refcd with fresh medium every ~ days. A suspension of mainly single cells was obtained from a 3-5-day culture by scraping or trypsinizing 1 the cell sheets off the glass and dispersing the cells with repeated pipetting. Cells grown in fluid suspension were also used suc- cessfully. In either case, the cell suspension was diluted in fresh medium to a final concentration of 10,000-15,000 cells/ ml based on a hemocytometer cell count at about the ~00,O00 cells/ml level. The cell suspension was placed in a 500-ml. Erlenmeyer flask equipped with a suspended Teflon-coated magnet and with glass and rubber tubing connections leading to a 1-ml. Cornwall syringe (Chart 1). A 3-way adapter placed

10.05-0.1 per cent trypsin (Difco 1:~50) in Hanks' salt solution minus calcium, magnesium, or phosphate.

959

Research. on May 14, 2020. © 1959 American Association for Cancercancerres.aacrjournals.org Downloaded from

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960 Cancer Research Vol. 19, October, 1959

on the syringe allowed recireulation of the medium that remained in the tubing between samples. The suspension was stirred continuously by a low-speed magnetic stirrer.

Preparation and testing of solutions.--Soluble test compounds were dissolved in distilled water or Hanks' salt solution with, in some cases, addition of small amounts of 0.5 N HC1, 0.5 N NaOH, ethanol, acetone, or methanol to promote solubility. Adjustment to pH 7.2-7.4 was then made when necessary with 0.5 N HC1 or 0.5 N NaOH. All solutions and broth filtrates were sterilized by UF-porosity glass filtration. Since only a small quantity of sample was needed for testing, it was convenient to use a 2-ml. Buchner-type UF glass filter flared at the top and seated in a standard l~-ml, centrifuge tube with a rubber O-ring placed under the flare to prevent glass-to-glass contact (Chart 1). Centrifugation for 15 minutes at 2000 r.p.m, was generally sufficient to obtain 1-1.5 ml. of sterile sample.

The culture containers used were the disposable 1.5-ml. vinyl plastic cup trays described by Rightsel and his co-workers (10)

growth medium to give the four or more desired test dilutions. HeLa cell suspension at 100,000-150,000 cells/ml in growth medium was added to each dilution at 10 per cent of final volume and thoroughly mixed by repeated pipetting. Two plastic cups were filled by pipette with 1 ml. for each of the test dilutions. As previously, the trays were covered with 2-inch strips of cellophane tape and incubated at 37 ~ C.

Cytotoxicity rating system.--All assays were ex- amined after 5 days' incubation for evidence of cytotoxicity. Cytotoxicity was judged by direct microscopic examination of the cultures at low- power, 63-100 magnification. The inhibition of cell metabolism as indicated by the phenol red color changes in the growth medium was taken as a secondary confirmation of cytotoxicity.

~r~,,.~., ,,,.,~, ;

1 F

FILTER

CHART 1.--Schematic diagram of experimental equipment

as being suitable for cell cultures. The trays were prepared as suggested by immersion in 95 per cent denatured alcohol for �89 hour and were dried in the bacteriological hood under UV light. The sterile test sample was dispensed directly into the plastic cups in graded volumes with a syringe mieroburet ~ and syringes calibrated at 0.2 ~d/unit of the microburet dial. A 30-gauge blunt-tip needle attached to the syringe allowed accurate placement of the aliquots into the cups (Chart 1). Cell suspension was then added to each cup to a total volume of 1 ml. For routine testing, a half-log dilution schedule was used with dilutions of 1/10, 1/32, 1/100, and 1/320.

Two cups were filled per dilution. A distilled-water control and a known-positive control were run with each test series. The plastic trays were covered with ~-ineh strips of Scotch cellophane tape with care taken to form an air-tight seal around each cup. Incubation of the cultures was at 37 ~ C.

Testing of insoluble compounds.--Insoluble compounds were suspended in a small amount of ethanol for �89 hour prior to the addition of sterile water or Hanks' salt solution to final volume. The sterile suspension was serially diluted in test tubes in

2 Micro-Metric Instrument Co., Cleveland, Ohio.

For microscopic evaluation of cell damage, art arbitrary scale of increasing cytotoxicity based on morphological characteristics was employed.

The main criteria for the rating system were:

Grade Definition 0 Cells firmly adhering to the plastic with clear details, and

forming a sheet-like monolayer. 1 Cell growth inhibited, but majority of cells adhering to

the plastic; some granulation and rounding up of cells. 2 Cells suspended and generally clumped, with pronounced

granularity and loss of cell detail; increasing cellular debris.

3 Mainly single, suspended, shrunken cells with irregular membranes; some disintegrating cell dusters and consid- erable cellular debris.

4 Complete cytolysis; all debris.

Determination of end-points.--Two e n d - p o i n t s w e r e d e t e r m i n e d fo r e a c h a s s a y : a cytotoxic end- point ( C E ) , o r c o n c e n t r a t i o n of d r u g c a u s i n g sig-



ToPLI~r--Tissue Culture Test for Large-Scale Screening 961

nificant cellular degeneration; and a lethal end- point (LE), or concentration causing complete cell disintegration. In terms of the cytotoxicity scale, the CE was the lowest concentration of drug that resulted in a rating greater than one scale unit over the control culture rating. With the control cultures of acceptable assays rated at 0 to �89 on the scale, the CE (1~J--s rating) was generally charac- terized by rounding and clumping of the cells in the medium with granulation, loss of cellular detail, and increased cellular debris. The LE was taken as the lowest concentration of drug giving a 3~-4 rating.

After 3-5 days' incubation, the growth medium of the cell cultures that contained lethal or cytotoxic concentrations of test substance usually, though not always, remained at the original pH of the medium (pH 7.~-7.4) or drifted to a slightly more alkaline condition, as shown by the phenol red indicator. Inactive substances did not prevent the normal change in medium pH toward the acid range during the incubation period. The cytotoxic activity as determined by this pH effect was re- corded as (q-) for the text substance if a definite inhibition of acid production was observed in any of the test dilutions; (+) if this effect was slight; and ( - ) if the dilution series indicated no inhibition of acid production as compared with the control cultures.

In the screening of fermentation broths, it was observed that some substances manifested cytotoxic activity only after 8-4 days' incubation. During the interval the cultures metabolized vigorously, and as a result, pH observations were a poor guide to cytotoxic concentrations. There- fore, microscopic examination of test cultures was, in all cases, the prime means of judging cytotoxicity.

Staining of cultures.--In addition to the direct rating of cell damage, staining of the test cultures for detailed study was possible by insertion of sterile l~-mm, circular glass coverslips into the plastic cups before an assay series was begun. The cells rapidly adhered to the glass bottom formed by the coverslips. The coverslips were easily removed for staining when desired.

RESULTS AND DISCUSSION A group of compounds of known tumor activity

have been tested by this technic, and the results are summarized in Table 1. a The cytotoxic end- point, lethal end-point, and activity by pH are given for each substance after 5 days' drug-cell contact. Also presented in Table 1 for direct com-

a Many of the compounds were generously supplied by the CCNSC.

parison are the available end-points for the same substances taken from the published data of Eagle and Foley (~-4).

In addition, plots of cytotoxicity scale rating versus drug concentration are presented in Chart

for a selected number of compounds. The plots illustrate the types of response curves obtained with a variety of cytotoxic drugs. The responses varied from compounds with a narrow range between the cytotoxic and lethal concentrations, e.g., puromycin and thioTEPA, to compounds with nonlethal cytotoxic activity over a broad concentration range, e.g., amethopterin and 6-mercaptopurine.

In their studies, Eagle and Foley determined the IDa0 for each drug, or concentration at which 50 per cent inhibition of growth was achieved in 5-8 days of drug-cell contact with protein analysis use(t as the measure of culture growth. The criteria for cytotoxicity described above require a more destructive effect on cell cultures beyond growth inhibition. Therefore, in general, the cytotoxic end-points determined by the methods described in this report require higher drug concentrations than the ID60's of Eagle and Foley. The end-points for compounds with sharply defined cytotoxicity, such as puromycin and thioTEPA, are in closest agreement with the IDs0's reported for these compounds by Eagle and Foley. The ratio of lethal to cytotoxic concentration for these compounds is generally between 5 and 10. In the case of compounds with nonlethal cytotoxic activity over a broad concentration range, such as amethopterin and 6-mercaptopurine, the two methods diverge with respect to end-point values. However, since both systems employ the same cell lines, growth medium, and approximately the same drug-cell contact times, the good over-all agreement as to the cytotoxic character of the compounds tested by both methods is not un- expected.

The morphological changes in the cell cultures upon which the cytotoxicity rating scale is based are the progressive symptoms of cellular degeneration most frequently observed with cytotoxic substances. Nitta (8), in studies of the action of anti- tumor antibiotics on HeLa cells, also noted that the various agents at suitable concentrations pro- duced common degenerative effects. However, the cytotoxic action of certain agents tested in our laboratory could not be categorized by the cytotoxicity scale. Certain drugs strongly inhibited cell growth at concentrations far below those that resulted in the common cytotoxic effects. The cell cultures appeared "frozen" on the plastic surface, although cell growth and metabolism, as indicated



TABLE 1

CYTOTOXICITY OF VARIOUS KNOWN COMPOUNDS

NSC no.

4 145 185 423 429 437 441 552 739 740 742 743 744 746 747 748 749 752 753 754 755 756 758 759 761 762 763 805

1112 1478 1481 1532 1571 1578 1589

1873

9-03O 2O89 9059 9O92 2100 2107 2359 ~432 2635 2652 2675 2773 2852 2853 3051 3053 3054 3055 3057 3060 .~ 3067 3075

3076

3081

3083

3087 3089

Name

2 -Amino -5 -nitrothiazole Phenylphosphonic acid Aetidione 2-(2,4-Dichlorophenoxy)ethanol 1,3-Diethylurea 3-Mereaptopropionie acid 2-Amino-$-methylJ-propanol 3-Methoxypropylamine Aminopterin Amethopterin Azaserine 2,6-Diaminopurine 6-Chloropurine Urethan

Cytotoxic end-point

(#g/ml) >60

> 100 0.5--3

20-100 > 100 > 100 > 100 > 100 1-10 1-5

O. 3-2 7-20

> 100 3200-10,000

Lethal end-point

(~g/ml) >60

>100 30-60

100-200 > 100 > 100 > 100 > 100

>50 > 100 2-10

20-60 > 100

_>__ 10,000

Activity by pH

n

+

+ ,_+ + ,_+

+ +

+ N-Methyl acetamide Formamide 8-Azaguanine 6-Thioguanine Purine B-2-Thienylalanine 6-Mereaptopurine 8-Azaxanthine D-Glueosamine hydrochloride Benzimidazole Methyl 3-pyridyl ketone (3-acetyl pyridine) Nitrogen mustard Dimethyl sulfoxide Meeonic acid (3-hydroxy-4-oxo-4-H-pyran-

2,6-dicarboxylic acid) Cyclopropaneearboxylic acid N-Butyl thiocyanate N-Lauryl thiocyanate 2,4-Dinitrophenol Resorcinol 3-Pyridinesulfonic acid, sodium salt 5-Pyrimidineearboxylic acid, 1,2,3,4-tetra-

hydro-2,4-dioxomonohydrate p-Acetamino-m-anisidine 2-Hydroxy-4,6-dimethyl pyrimidine hydro-

chloride 8-Hydroxyquinoline fl-Diethylaminoethyl chloride hydrochloride B-Benzoylpropionie acid Nitrofurazone Furadantin N-Phenylsuccinimide 2,4'-Sulfonyldiphenol Methyl chloroacetate 2-Dimethylaminoethanol Tri-n-butyl phosphite N-2-Hydroxyethylphthalimide 2-Naphthoic acid, 3-hydroxy-7-sulfo- Benzoic acid, m-sulfo- N-Methyl formamide Actinomycin D Methyl carbamate Puromycin dihydrochloride 8-Aza-2,6-diaminopurine sulfate Potassium arsenite Daraprim, pyrimethamine Netropsin hydrochloride 4,6-Diamino-l-(p-carboxyphenyl)-l,2-dihy-

dro-2,2-dimethyl-s-triazine, hydrochloride 4-Amino-6-anilino-l,2-dihydro-2-phenyl-s-

triazine 4,6-Diamino-l-(4'-chlorophenyl)-2-n-hexyl-

1,2<lihydro-s-triazine hydrochloride 4,6-Diamino-l-(8'-chlorophenyl)-l,2-dihy-

dro-2-n-hexyl-s-triazine hydrochloride Oxophenarsine hydrochloride Methylene blue chloride

2400-7500 > 1000

2-10 2-10 6-2O

380-1200 20-100

>6O > 100 > 100 > 100 1-5

> 100

> 100 > 100 20-100

6-40 20-40

320-1000 > 100

>6O > 100

> 100 0.2-1

10-60 > 100 7--60

10--60 > 100 2O-60 10-30 > 100 > 100 > 100 > 100 > 100 > 100

0.01-0.04 > 100

0 .1-0 .5 >60 3-10

2O--60 30-100

>60

6-20

10-60

13-40 6-20 1-3

> 7500 > 1000 40-100 60-100 60--200

1200-3800 > 100

>60 > 100 > 100 > 100 5-20

> 100

> 100 > 100

100-400 40-120 >200

1600-10,000 > 100

>60 > 100

> 100 O. 7-2

60-200 > 100

> 60 100-200

> 100 >60

30-200 >100 > 100 > 100 > 100 >100 > 100

0.1--0.5 > 100

0.5-2 >6O

10-20 >6O

100-300

>6O

>2OO

60-200

60-200 30-100 10-30

+

+ + + + +

+ m

+ +

+ , + +

m

+ +

+ +

_+ +

+

+

+

+

+

+

+ +

?

Eagle & Foley* ID6o

(.g/toO > 100 > 100

O. 1-1 1-10

> 100 >100 > 100 > 100

O. 01--0.1 0.01-0.1

0.1-1 1-10

10-100 > 100 > 100 > 100

O. 1-1 0.1-1 0.1-1

> 100 0.1-1

10-100 > 100 >100 > 100

O. 1-1 > 100

> 100 > 100 > 100

O. 1-1 10-100 >100 > 100

> 100 > 100

> 100 O. 1-1

1-10 > 100 1-10

>IO0 >100 10-100 10-100 > 100

>10 > 100 > 100 > 100 > 100

O. 01-4). 1 > 100

O. 1-1 > 100

0 . ] -1 0.1-1

10-100

> 100

O. 1-1

1-10

O. 1-1 1-10

O. 1-1

* References 2, 3, 4.

962



TABLE 1--Continued

NSC no. Name Cytotoxic Lethal Activity Eagle & Foley* end-point end-point by pH IDs0

8090 3091 $092 3093 3206 4240 5036 5201 5205

5206

6896 9706

10107 19645 19893 20200

26980

Crystal violet Methyl green Neutral red Pyronin B S-Benzylthiuronium chloride 4-(p-Diethylaminostyryl)quinoline Caffeine p-Biguanidobenzamide 4,6-Diamino-l,2-dihydro-2,2-dimethyl-1-

(2,6-xylyl)-s-triazine hydrochloride 4,6-Diamino-l-(m-bromophenyl)-l,2-dihy-

dro-2-(n-undecyl)-s-triazine hydrochloride ThioTEPA Triethylenemelamine Nitromin 4-Nitroquinoline-l-oxide 5-Fluorouraeil Benzyldimethylammonium chloride mono-

hydrate Mitomycin C

(ttg/ml) 0.3-1

20-100 20-60

2-6 10-30

3-20 > 100

>60

> 100

7-20 5-20 1-5 6-20

0.03-4). 1 6-20

6-20 0.1-0.2

(~g/ml) 3-6

100-200 100-200

10-30 30-200 30-200 >100

>6O

>100

20-30 20-100

5-20 20-60

0.2-0.6 2100

30-100 0.5-2

? ? ? ? + +

+ + + + + +

(~tg/ml) 0.001-0.01

10-100 1-10

0.1-1 0.1-1 0.1-1

> 100 > 100

>100

1-10 1-10

TABLE 1: ADDENDUM

CYTOTOXICITY OF VARIOUS KNOWN COMPOUNDS

(Additions and revisions as of September 1, 1959)

NSC no. Name Cytotoxic Lethal Activity Eagle & Foley end-point end-point by pH IDs0

-

643 741 750

2105 3056 3058 8069 3073 3085 3095 3099 4239

4730 5204

5366 26271

Benzenesulfonhydrazide Hydroeortisone Myleran Isopropyl N-phenylcarbamate Puromycin aminonucleoside Benzotriazole

(#g/ml) > 100 40-120

160-600 500-1600 100-500 160-400

(#g/mD >100 2400 ~600

1600-5000 >500

1200-5000

+ , + + , + + , + +,_+

+ Chloramphenicol Folie acid DL-Desthiobiotin 1,5-Diaminobiuret Galactoflavin 4-(p-Dimethylaminostyryl)quinoline

methiodide 2-Ethylamino-l,3,4-thiadiazole 4-Amino-6-anilino-l,2-dihydro-2,2-dimethyl-

s-triazine Narcotine Cytoxan

> 100 > 1000 > 1000 80-250

250-1600

6-40 300-940

200-780 8-50

>200

>100 >1000 > 1000

2500 1600-5000

40-120 1500-3000

1000-2500 80-250 >200

+

+ +

+,_+ +

(V:g/ml) 10-100

> 100 > 100

>10 1-10 > 100

10-100 > 100 > 100

10-100 > 100

0.1-1 > 100

> 100 1-10

963



964 Cancer Research Vol. 19, October, 1959

by acid production, had ceased. For these and any other cultures over which there was any doubt concerning viability, a recovery step was added to the test procedure. After 5 days' incubation, the drug solution was removed, and fresh growth medium was added to the cell cultures. The cups were resealed and incubated for an additional 3-5 days. Microscopic examination of the cultures for the presence or absence of actively multiplying cells allowed accurate pinpointing of lethal drug concentrations.

The tissue culture test described in this report is currently used in our laboratory as an auxiliary

(.D Z l--

rl-

>- t-- 0 X 0 I-- 0 I-- >.-

4

Puromycin.2HC!

=,=

= 1

0 . 0 5 0 . 5 5

screen to the experimental animal tumors for the detection of potential anti-tumor substances. I t has proved to be a rapid, inexpensive, and re- producible method for determining the eytotoxici- ty of large numbers of samples. A well trained technician can easily perform and evaluate 80 assays daily; that is, 80 samples at four dilutions in duplicate. This includes the routine mainte- nance of stock cultures and other associated duties. In addition, the test serves as an assay method for anti-tumor activity that is a valuable aid in the fractionation of crude preparations that show experimental tumor and cell culture activities. Tis-

Thi0-TEPA

i

�9 �9 i * * a i , , i i �9 I , l l l | |

I0 I 0 0

6-Mercaptopurine I A-Methopterin

I0 I00 i I0 I00

C O N C E N T R A T I O N IN p g / m l

CHART 2.--Curves showing cytotoxicity scale rating as a The readings were made on HeLa cell cultures after 5 days function of drug concentration for four selected compounds, incubation at 37 ~ C.

FI~. 1-5.--Photomicrographs of HeLa cells showing typi- cal appearance of cultures for each grade of the cytotoxicity rating scale. X200. The grades for the figures are 0, 1, 2, 3, and 4, respectively.



ToPLIN- -T i s sue Culture Test for Large-Scale Screening 965

sue cu l tu re resul ts are c o n s t a n t l y m o n i t o r e d by t h e an ima l t u m o r tests. I t has been our exper ience t h a t cell cu l tu re a c t i v i t y is a re l iable measu r e of t he degree of c o n c e n t r a t i o n ach i eved in a chemica l o p e r a t i o n on a cy to tox i c a n t i - t u m o r b ro th f i l t ra te . I n m a n y cases, a p r e l i m i n a r y e v a l u a t i o n of cy to- toxic a c t i v i t y a f t e r on ly 3 days ' i n c u b a t i o n is suffi- c i en t for d e t e r m i n i n g t he effect iveness of a par- t i cu l a r t r e a t m e n t .

S U M M A R Y

A simple, rap id , a n d r e l a t i ve ly inexpens ive tissue cu l tu r e c y t o t o x i c i t y t e s t su i tab le for large- scale cancer c h e m o t h e r a p y sc reen ing has been de- scr ibed. T h e m e t h o d invo lves t h e microscopic e v a l u a t i o n of c y t o t o x i c i t y aga ins t h u m a n cell cul- tu res for v a r y i n g d i lu t ions of t e s t subs t ance a f t e r 5 days ' i ncuba t ion . T h e resul t s o b t a i n e d b y th i s t e c h n i c wi th a g roup of k n o w n c o m p o u n d s h a v e been given. Some p r e l i m i n a r y obse rva t i ons h a v e been m a d e on t h e app l i ca t ions a n d l i m i t a t i o n s of t h e m e t h o d in r a n d o m screening a n d in fo l lowing t h e c o n c e n t r a t i o n of a n t i - t u m o r agen t s f rom c rude p r epa ra t i ons .

ACKNOWLEDGMENTS I would like to express my thanks to Dr. Frank M. Schabel,

Jr., of the Southern Research Institute for his original sugges- tions on the practicality of a cytotoxicity test of this type in cancer chemotherapy screening, and to Mr. Ren6 Schmitter for technical assistance.

REFERENCES 1. EAGLE, H. Nutrition Needs of Mammalian Cells in Tissue

Culture. Science, 122: 501-4, 1955. $. EAGLE, H., and FOLEY, G. E. The Cytotoxic Action of

Carcinolytic Agents in Tissue Culture. Am. J. Med., Ill: 739-49, 1956.

3. ~ . Cytotoxicity in Human Cell Cultures as a Primary Screen for the Detection of Anti-Tumor Agents. Cancer Re- search, 18:1017-~5, 1958.

4. ~ . Susceptibility of Cultured Human Cells to Anti- Tumor Agents. Ann. N.Y. Acad. Sc., 76:534-41, 1958.

5. FOLEY, G. E.; EAGLE, H. ; SNELL, E. E.; KIDDER, G. W. ; and THAYER, P. S. Studies on the Use of In Vitro Procedures for the Screening of Potential Anti-Tumor Agents: Com- parison of Activity in Mammalian Cell Cultures and Micro- biological Assays Alone and in Combination with Experi- mental Anti-Tumor Activity. Ann. N.Y. Acad. Sc., 76: 952- 60, 1958.

6. HmSCHBEnO, E. Tissue Culture in Cancer Chemotherapy Screening. Cancer Research, 18: 869-78, 1958.

7. NITTA, K. Studies on the Effects of Aetinomycetes Prod- ucts on the Culture of Human Carcinoma Cells (Strain HeLa). I. The Effect of Known Antibiotics Having No or Slight Tumor-Inhibitory Activity on HeLa Cells. Jap. J. Med. Sc. & Biol., 10:$77-86, 1957.

8. ~ . Studies on the Effects of Actinomycetes Products on the Culture of Human Carcinoma Cells (Strain HeLa). II. The Effect of Known Anti-Tumor Antibiotics on HeLa Cells. Ibid., pp. $87-96.

9. ~ . Studies on the Effects of Actinomycetes Products on the Culture of Human Carcinoma Cells (Strain HeLa). III. Comparative Studies on Anti-HeLa-Cell Effects of Known Synthetic Anti-Tumor Substances. Ibid., pp. 419- $8.

10. RIGHTSEL, W. A.; SCHULTZ, P.; MUETHING, D.; and McLEAN, Ja., I. W. Use of Vinyl Plastic Containers in Tis- sue Culture for Virus Assays. J. Immunol., 76:464-74, 1956.



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