tests for collateral sensitivity or cross resistance...
TRANSCRIPT
TESTS FOR COLLATERAL SENSITIVITY OR CROSS
RESISTANCE IN BACTERIAL MUTANTS RESISTANT
TO AMETHOPTERIN OR FURINE ANALOGS*
Robert Guthrie, Maude E. Loebeck,Marilyn J. Hillman and Franiszek Zgorzynski
Roswell Park Memorial InstituteNew York State Department of Health
Buffalo, New York
Law (1)reported that certain lines of leukemia in mice became more
sensitive to amethopterin when resistance to 6-mercaptopurine (6-MP) devel
oped, or when resistant to, or "dependent" upon, 8-azaguanine (8-AG). Szy-
balski and Bryson (2) observed an instance of mutation in Escherichia coli
wherein increased resistance to one antibiotic was associated with increased
sensitivity to a second. They designated this phenomenon "collateral sensitiv
ity". Subsequently, Elion, et al. (3) found a 2,6-diaminopurine (DAP)-resis-
tant strain of Lactobacillus casei that possessed increased sensitivity to 6-MP.
Elion and Hitchings (4) reported a 6-MP-resistant strain of L. casei with in
creased sensitivity to amethopterin and conversely Hutchison and Burchenal
(5)observed collateral sensitivity to 6-MP in an amethopterin-resistant Strep
tococcus fecalis strain. Schabel, Wheeler and Skipper (6) found that an aza-
serine-resistant strain of _E. coli was more sensitive than the parent to 6-MP
and diaminobiuret, and that a sulfanilamide-resistant strain showed collateral
sensitivity to 6-chloropurine, 8-AG, or purine.
In our laboratory, mutants of _E_.coli resistant to purine antagonists
and mutants of Bacillus subtilis resistant to amethopterin have been used to
*This work was supported, in part, by a research grant (C-2593) from the
U. S. Public Health Service.
319
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320 Cancer Research Guthrie et al.
screen compounds for occurrence of either cross-resistance or collateral
sensitivity to these compounds. To date, collateral sensitivity has been dem
onstrated to amethopterin in three E. coli mutants resistant to purine antagon
ists. Collateral sensitivity to certain analogs of the thiamine pyrimidine has
been found in amethopterin-resistant B. subtilis mutants, as well as the
E. coli mutants mentioned above. For one thiamine pyrimidine analog, 2-
hydroxy-4-amino-5-aminomethyl pyrimidine, cross-resistance in B. subtilis
amethopterin-resistant mutants was found, while E. coli mutants resistant to
purine analogs were collaterally sensitive to it (7).* One analog, 2-methyl -
mercapto-4-amino-5-hydroxymethyl pyrimidine, was subsequently tested in
mice and found to significantly retard a leukemia and a carcinoma (8). Nega
tive experiments, using this compound in rodent tumors, are reported else
where in this supplement (9). Compounds reported here are those for which
neither collateral sensitivity nor cross-resistance was demonstrated during
two or more separate tests comparing one or more amethopterin-resistant
mutants of B. subtilis with the parent strain, and one or more E. coli mutants
resistant to purine antagonists with the parent E. coli strain.
Several amethopterin-resistant mutants were isolated from popula
tions of the parent, Bacillus subtilis ATCC 6051, and from a purineless auxo-
troph, B. subtilis 6051-9 (10). Mutants resistant to 6-MP were isolated from
a purine-requiring E. coli strain, 9661-01 (10). Two mutant strains were
isolated from cultures of the 6-MP-resistant organisms which were resistant
simultaneously to 8-AG and 6-thioguanine (6-TG), as well as to 6-MP. Table I
lists a number of the strains used. In all cases, the drug-resistant mutants
were isolated from solid agar media by incorporating large populations of the
* Guthrie, R., Loebeck, M. E., and Hulmán, M. J., PROC. SOC. EXP.
BIOL. MED. (in press)
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Guthrie et al. CancerChemotherapyScreeningData 321
sensitive parent into minimal agar medium containing the drug, incubating,
and subculturing discrete colonies that appeared. Using these resistant strains
and the parent strains, 278 compounds were tested, including 182 pyrimidines
and 29 purines. Each compound was tested alone and also in combination with
amethopterin or 6-MP, to detect possible synergistic effects. If the amethop-
terinor purine analog-resistant mutant consistently showed significantly great
er sensitivity to the test compound than the parent in 3 or more replicate ex
periments, the resistant strain was considered to possess collateral sensitiv
ity to the test compound.
The agar-diffusion method was used for routine testing. Two dif
ferent minimal, chemically-defined agar media, one for each bacterial species
(10, 11) were homogeneously seeded with the test organism and distributed in
petri dishes. Each compound was tested by impregnating filter paper discs
(6.5 mm or 12.7 mm diameter) with solutions containing approximately O.l^fM.
or O.S^M, respectively, and placing them upon the surface of the agar. The
width of the inhibitory zone, if any, as well as completeness of inhibition with
in the zone, was noted. The resistant strains varied in their response to test
compounds, and the choice of those used in screening was arbitrary. There
fore, it is possible that other mutant strains of B. subtilis or of E. coli resis
tant to the same agents may demonstrate collateral sensitivity or cross-resis
tance to compounds reported here as negative with our mutant strains. B.
subtilis 6051, 6051/A-l, 6051/A-2, E. coli 9661-01 and 9661-01/668-1 strains
(see Table I) were used to test all but a few of the compounds reported here.
REFERENCES
1. Law, L. W., PROC. SOC. EXP. BIOL. MED., 78, 499 (1951).
2. Szybalski, W., and Bryson, V., J. BACT., 64:489 (1952).
Research. on January 16, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from
322 Cancer Research Guthrie et al.
3. Elion, G. B., Singer, S., Hitchings, G. W., Balis, M. E. and Brown,
G. B., J. BIOL. CHEM., 202:647 (1953).
4. - and Hitchings, G. W., PROC. AM. ASSN. CANCER RESEARCH,
1_:13 (1953).
5. Hutchison, D. and Bur chenal, J. H., Chemistry and Biology of Pteridines,
J. and A. CHUCHILL, LONDON, 1954, 366.
6. Schabel, F. M., Jr., Wheeler, G. P. and Skipper, H. E., PROC. AM.
ASSN. CANCER RES., 2:43, (1955).
7. Loebeck, M. and Guthrie, R., BACT. PROC., 1956, 121.
8. Guthrie, R., Holland, J. F., Hyatt, E., Hulmán, M., and Mount, D. T.,
PROC. AM. ASSN. CANCER RES., 2:113 (1956).
9. Holland, J. F., Guthrie, R., Tieckelmann, H. and Cuddihy, R., CANCER
RES. SUPPL. (in press).
10. Guthrie, R. and Lu, Wan Ching, BACT. PROC., 1953, 90.
11. - , J. BACT., 57:39 (1949).
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Guthrie et al. Cancer Chemotherapy Screening Data 323
TABLE I
MUTANT BACTERIAL STRAINS AVAILABLE FOR TESTING
Mutant Strain
1.2.3.4.5.6.7.8.9.10.11.12.13.14.15.16.B.subtilis 6051parent6051/A-l6051/A-26051/A-36051/A-46051/A-l/AG-l"
6051-9 purine-
requiringmutant6051-9/A-l6051-9/A-26051-9/A-36051-9/A-4E.
coli 9661-01purine-requiring
parent9661-01/MP-l9661-01/MP-29661-01/MP-59661-01/668-1
Increased Resistance to:
Amethopterin
Amethopterin, 8-azaguanine
Amethopterin
6- Mer captopurine
6-Mercaptopurine, 6-thio-guanine, 8-azaguanine
17. 9661-01/668-2
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324 Cancer Research Guthrie et al.
TABLE II
SOURCES
Code Source
A Aldrich Company
AC American Cyanamid Company
ART Prof. A. R. Todd
B Dr. C. T. Bahner
C Cyclo Chemical Corporation
CF California Foundation for Biochemical
ResearchE Eastman Kodak Company
EA Dr. E. P. Anderson
F Fisher Scientific Company
GBI General Biochemical, Inc.
H Dr. J. Hoover
IS Dr. Irving Slotnick
JS Dr. James Sprague
K Krishell Laboratories, Inc.L Eli Lilly CompanyM Merck and Company
NBC Nutritional Biochemical CompanyPD Parke, Davis Company
S Searle CompanySKI Sloan-Kettering Institute
SI Squibb Institute for Medical Research
V Dr. D. VisserWG Dr. William Garner
WRL Wellcome Research Laboratories
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1958;18:319-334. Cancer Res Robert Guthrie, Maude E. Loebeck, Marilyn J. Hillman, et al. Mutants Resistant to Amethopterin or Purine AnalogsTests for Collateral Sensitivity or Cross Resistance in Bacterial
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