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Effects of piposulfan (ancyte) on protein and DNA synthesis in ehrlich ascites carcinoma
Life Sciences Vol . 10, Part II, pp. 605-812, 1971 . Printed in Great Britain
Pergamon Press
EFFECTS OF PIPOSIII.FAN (ANCYTE) ON PROTEIN AND DNA SYNTHESIS IN EHRLICH ASCITES CARCINOMA Tobias 0 . Yellin* Dept . of Chaoical Pharmacology, Abbott T.aboratoriee, North Chicago,I11 .60064
(Received 1 March 1971; in final form 12 April 1971) summary
The effectn of the difuactional alkylating agent piposulfan on DNA, RNA and protein synthesis were studied in cell auspeasiona of Shrlich ancitas carcinoma derived from mice treated with the drug . Piposulfaa did not act like a typical alkylating agent . It iahibitad the incorporation of 3H - thymidine and markedly increased that of 14C -leucine without affecting thn labeling bq 3H uridine of TCA insoluble material . The effects of the drug were delayed fos about 16 hours .
Piponulfan (Ancyta) or N,N'-bie-(3-methylsulfonyloxy)-piperazine is a member of a narlen of neutral, difunctional alkylating agents studied at Abbott Laboratorien for their aatineoplantic activity (1,2) .
The first member of the
series, pipobroman (Vercyte~, is presently in clinical use for the treatment of polycythamia vera and chronic granulocytic leukemia . piponulfan sad pipobraman are nhown in Figure 1 .
The structures of
Like the parent compound,
piponulfaa (NSC 47774) showed a wide npactrum of antitumor activity in animals (3,4) sad has undergone clinical trials .
The results demonstrated activity
against lynphamas, chronic granulocytic leukemia, chronic lymphocytic leukemia and polycythemia vara (5) .
In this communication we report our initial studies
concerning the ponsible mode of action of piposulfan . The mechanism of action of alkylatiag agents is unknown (reviews, 6-9) . It in difficult to determine their primary site of action because they can react with a wide variety of nucleophiles within the cell including proteins * Prenant address :
Department of Pharmacology, Smith, Küne end French Laboratories, Philadelphia, Pe .
605
608
Effect oaf Alkylating Agents
Vol. 10, No . 11
and nucleic acids and, therefore, can cause many diverse biochemical lesions . An appropriate example is busulfan (Myleran) .
Like pipoaulfan, busulfan is a
difunctional alkylating agent of the alkylaulfonic eater type
(see Fig. 1) .
Buaulfan has been shown to alkylate proteins and nucleic acids (1 U) ; to be FIG . 1
I+ Il CH3-S-0-CHZ-CHZ-C-N, G 0 Pipoaulfan
~
II II N-C-CHZ-OS-CH3
(Ancyte)
CH3-S-0-CHZ-CHZ-CH Z-CHZ-O-S-CH3
li
II
0
0 Busulfan (Myleran®)
0 . ._ 0 u , Br-CHZ-CHZ-C-N, , ~N-C-CHZ-CHZ-Br Pipobruman (Vercyte ®) incorporated into DNA (11) ; to react with cyateine and other thiola in vivo (la) ; t~~ ini~ibit .)NA synthesis (13) ; to inhi~it mitosis and cause giant cell '.urination (14) ; to inhibit spermatogenesis in rata (15) ; to cause mutations in ~?rosophilia
(16) ; and to inhibit crystalline trioeephosphate dehydrogenase (17) .
Uf course we could expect a similar multiplicity of action with pipoaulfan and consequently difficulty in determining the mechanism of ite antitumor activity .
Nevertheless, it was of interest to question how it might be typical
or atypical of this class of drugs . Materials and Methods î,e11 Line Ehrlich ascitea carcinoma subline ES-III a'/357 was obtained from A.D . Little ~ Co ., Cambridge, Mass ., and maintained here by weekly transplant in
Effect od Alkylating Agents
Vol . 10, No . 11
807
Swiss-Webster male mice (25-35 gm) . Cell Couat and Viability Cell coasts ware done with the aid of a hemocytometer .
Viability was
calculated from the total number of cells minus the number of cells staining with Trypaa Blue (0 .07x) divided by the total number of cells . Assay of Macramolecular Synthesis At appropriate times, cell were removed from the peritoneal cavity and washed in Eagle's M~I, pH 7 .
Five to ten million cells from each animal were
suspended, in duplicate, in two mls of Eagle's MEM (Hank's salts) with 2mM glutamine, pH 7 .2, containing radioactive precursor and incubated for 1 hour at 37 ° C in air, with shaking .
Protein, RNA and DNA synthesis was measured
as TCA insoluble radioactivity from uridine (500 mCi/mmol) sad
14 C - leucine (311 mCi/mmol),
3H -
3fi -thymidine (500 mCi/mmol) respectively .
The
reaction was stopped with 3 ml of Sx TCA containing 1 mg/ml cold precursor and the mixture centrifuged .
The cells were washed again is TCA, then in
ethanol and twice more is athaaol :ether (3 :1) .
The residue was dried in a
water bath, dissolved in 0 .5 ml formic acid and the radioactivity determined in liquid scintillation spectrometer . Results and Discussion Table 1 gives the results of a representative experiment designed to teat the effects of piposulfaa on DNA, RNA and protein synthesis is vivo .
Seven
days after transplaatatioa (106 cells), six mice were randomized into two equal groups .
Group C received two iatraperitoneal (i .p .) injections of 0 .5
ml saline and group X vas given two injections of 100 mg/kg of pipoaulfan i .p . in saline seven hours apart .
The aaimala were sacrificed 24 hours after the
first treatment sad the tumor cells processed as described under Materials sad Methods .
The data show a 48S increase in the rata of protein synthesis,
no significant change in. RNA synthesis and a 25x decrease in the rate of DNA synthesis in carcinoma cells from animals treated with piposulfan .
Evidently
these effects would precede a "clinical" response to pipoaulfan since neither
808
Effect oaf Alkylating Agents
Yol . 10, No. 11
cell viability nor cell count was significantly altered bq the drug at this time .
Routine microscopic examination showed no noticeable difference in
appearance between control and experimental calls . A similar pattern of activity was found in preliminary experiments using the secitea form of the 4lalker 256 sarcoma is rate . TABLE 1 Effects of Piposulfan on Macromolecular Synthesis and Cell Count in Ehrlich Asc hes Carcinoma After One Day . of Treatment Total Cell Count Lilliona~
Cell Viability
Mouse
_TCA Insol . Radioactivity (CPM) 1 Leuçine Uridine Th~m idine
C1
17,243
82,289
22,849
225
98
C2
14,722
80,736
22,068
400
90
C3
14,485
88,641
17,940
190
95
Average 2 15,480
83,922
20,952
272
94
± 879
*- 2407
~ 1523
X1
27,508
83,765
17,488
189
95
X2
23,785
80,014
17,645
204
95
X3
17,885
94,557
15,792
330
90
Average
23,059
86,109
15,792
241
93
t 2802
± 4360
± 1775
148
102
75
89
99
~ of Control
7
l Cell number was identical in all tubes,. 2 Averages are given ± S .E . Table 2 shows the results of an experiment similar to the one above except that the tumors were treated for two days (2 x 100 mg/kg/day) . Here we observe pronounced effects on protein and DNA synthesis again without
Vol . 10, No. 11
Effect ad Alkylating Agents
significant change in ßNA synthesis .
609
Dote also the marked reduction in cell
count indicating a therapeutic response to piposulfan .
Since viability re-
mains unchanged at this time, ae might surmise that mitosis has been arrested by the drug . cell volume
Consistent with this, ae observed a definite lncreaae in the (giant cells) of the carcinoma cells from treated animals after
tao days . TABLE 2 ßffecte of Piposulfan on Macromolecular 5yntheaia and Cell Count in Ehrlich Asches Carcinoma After ~o Days of Treatment Total Cell Count (millions)
Cell Viability X
22,537
189
95
95,527
21,232
180
86
19,587
72,437
24,599
105
98
Average2 19,357
86,984
22,786
158
93
t 373
t 7311
t 980
X1
29,.915
66,522
13,111
39
100
XZ
53,983
95,456
17,012
60
94
X3
40,177
76,924
15,118
39
93
Average
41,358
79,634
15,080
46
93
t 6973
t 8462
± 1126
213
91
66
29
103
Mouse
TCA Insol . ßadioactivity (CPM)1 Laudas Uridino Thymidine
C1
19,857
92,988
C2
18,628
C3
X of Control
1 Cell number ass identical in all tubes. 2 Averages are given t S .ß . Table 3 sussarises additional experiments similar to the ones described above .
It may be seen that the effects of piposulfaa on protein sad DdiA syn-
thesis became evident betaean 16 and 24 hours after drug administration and
810
Effect ad Alkylating Agents
becona pronauad at later times .
Vol. 10, No . 11
We do not know if the drug affects the syn-
thasis of DNA sad protein independently of each other .
It would be interesting
to learn what effect piposulfan may have on protein synthesis in systems where nucleic acid synthesis is not present, as in reticulocytes, for example . TABLE 3 Effects of Pipoaulfan on Labeling of DNA and Protein
Bty. #1 1
(3)
2
(6)
3
Treatmant2 (nft/ka i .p .)
Time of Sacrifica3 (Hours)
504
Incorporation of Precursor as of Control ThS+midine Leucine
x
16
90
114
2 x 25
24
89
142
(6)
2 x 50
24
77
149
4
(3)
50
40
73
196
5
(3)
2 x 50
48
56
296
1 The number of mice per group is given in parentheses . 2 Where two injections were given they were seven hours apart on the first day of treatment, 3 Time given from the first injection. 4 The i .p . LD50 of piposulfan in Swiss-Webster mice is 400 mg/kg (4) . Table 4 gives the results of experiments in which DNA synthesis was measured 2 and 4 hours after a single intraperitoneal dose of 200 mg/kg of pipoaulfan or pipobroman (3 animals/group) .
Pipobraman, which seems to act
like a "typical" alkylating agent (see below), differs from piposulfan in having bramiaa instead of methylsulfonic acid as the leaving group in reactions with nuclaophilas (see Figure 1) and it is interesting to note that this makes quite a difference biologically .
In contrast to drastic inhibition of DNA
synthesis by pipobrae~an, piposulfaa had no effect at 2 and 4 hours after drug adniaistration .
Neither drug affected protein synthesis at these times but
pipobranan slightly inhibited (ca, 25x) RNA synthesis at 4 hours.
Vol. 10, No . 11
Effect ad Alkylating Agents
611
TABLE 4 Comparison of the Effect of Piposulfaa sad Pipobroman on DNA Syathesis l Two sad Four Hours After Drug Administration
Sam~e2
Time of Sacrifice
Average Radioactivity t S~E~ ~PM)
Relative Açtivity
Controle
2
12805 t 867
100
Piposulfan
2
13809 t 451
108
Pipobroman
2
2629 t 360
21
Controls
4
8773 t 1136
100
Piposulfan
4
9301 t 487
106
Pipobroman
4
1770 t 367
20
1 Incorporation of 3H-thymidine into TCA insoluble material . 2 Each group had three mice . 3 Cell number Was identical in all tubes . Among other possibilities, such se inhibition of the biosynthesis of a vital cell component and its subsequent exhaustion from the cell, the delayed onset of activity with pipoaulfan may indicate a site of action which ie available during only part of the cell cycle .
Future experiments using
synchronized cultures could prove useful to our understanding of its mode of action and of cell biology .
Its effect on protein syatheeis merits further
study in itself and here also pipoaulfan may prove to be a useful biochemical tool . References 1.
J . A . CARBON, S . M . BREM aàd J . D, RATAJCZYK, Abstr . Paper, 139th Meeting, Am . Chem . Soc . (1961) .
2.
R, J . STEIN, J . A . CARBON, J . LANGDON and R, K, RICHARDS, J . Lab . Clin . Med . 5 6 :949 (1960) .
3.
Sloan-KetterinA Tumor Spectrum Studies , SK#24591, (1961) .
4.
T . J, MCNAIR, F . A, WIBIN, E . T, HOPPE, J, L . SCHMIDT and F, A, dePEYSTER, J . S4rft . Res . _, 130 (1963) .
612
Effect of Alkylating Agents
Vol. 10, No . 11
5.
N. A, NELSON, R, W. TALLEY, M. L. REED, A, M, EVANS, B. L . ISAACS, P. RUFFItAN and J. LOUIS, Clin . Pharmacol, Therau . 8, 385 (1967) .
6.
A. HADDOW ;
7.
G, P, WHEELER,
Cancer Rea .
22, 651 (1962) .
8,
G, P, WARWICK,
Cancer Rea,
23, 1315 (1963) .
9.
H. VERMIAZD and F . F, COLL7N,
10 .
E, G. TRAMS, M, V . NADRARNI, V. DEQUATTRO, D, MAStiGWYN-DAVIES and P, K. SMITH, Biochem, Pharmacol. 2, 7 (1959) .
Physiopatholomr of Cancer , p . 602, N, Y., Hoeber Prese (1959) .
Cancer
21, 58 (1968) .
11 .
E, G. TRAMS, M. V, NADKARNI and P. K. SMITH,
12 .
J . J. ROBERTS and G. P. WARWICK, Biochem. Pha:macol .
13 .
J. G. LI, M. E, LEONARD and G . HARRISON,
14 .
R, BASSLEER,
15 .
K. A. AHLQUIST,
16 .
0, G. FAHM1f and J, J. FAHMY,
17 .
H. HOLZER,
Comet . Read . Soc . Biol . J. Reurod . Fart .
Cancer Rea.
Cancer
21, 560 (1961) .
6, 216 (1961 ; .
9, 963 (1956) .
~, 237 (1965) .
12, 377 (1966) .
J. Genetics
46, 361 (1961) .
Die Mediziniache , 576 (1956) ; CA 50, 10257 (1966) .
Pergamon Press
EFFECTS OF PIPOSIII.FAN (ANCYTE) ON PROTEIN AND DNA SYNTHESIS IN EHRLICH ASCITES CARCINOMA Tobias 0 . Yellin* Dept . of Chaoical Pharmacology, Abbott T.aboratoriee, North Chicago,I11 .60064
(Received 1 March 1971; in final form 12 April 1971) summary
The effectn of the difuactional alkylating agent piposulfan on DNA, RNA and protein synthesis were studied in cell auspeasiona of Shrlich ancitas carcinoma derived from mice treated with the drug . Piposulfaa did not act like a typical alkylating agent . It iahibitad the incorporation of 3H - thymidine and markedly increased that of 14C -leucine without affecting thn labeling bq 3H uridine of TCA insoluble material . The effects of the drug were delayed fos about 16 hours .
Piponulfan (Ancyta) or N,N'-bie-(3-methylsulfonyloxy)-piperazine is a member of a narlen of neutral, difunctional alkylating agents studied at Abbott Laboratorien for their aatineoplantic activity (1,2) .
The first member of the
series, pipobroman (Vercyte~, is presently in clinical use for the treatment of polycythamia vera and chronic granulocytic leukemia . piponulfan sad pipobraman are nhown in Figure 1 .
The structures of
Like the parent compound,
piponulfaa (NSC 47774) showed a wide npactrum of antitumor activity in animals (3,4) sad has undergone clinical trials .
The results demonstrated activity
against lynphamas, chronic granulocytic leukemia, chronic lymphocytic leukemia and polycythemia vara (5) .
In this communication we report our initial studies
concerning the ponsible mode of action of piposulfan . The mechanism of action of alkylatiag agents is unknown (reviews, 6-9) . It in difficult to determine their primary site of action because they can react with a wide variety of nucleophiles within the cell including proteins * Prenant address :
Department of Pharmacology, Smith, Küne end French Laboratories, Philadelphia, Pe .
605
608
Effect oaf Alkylating Agents
Vol. 10, No . 11
and nucleic acids and, therefore, can cause many diverse biochemical lesions . An appropriate example is busulfan (Myleran) .
Like pipoaulfan, busulfan is a
difunctional alkylating agent of the alkylaulfonic eater type
(see Fig. 1) .
Buaulfan has been shown to alkylate proteins and nucleic acids (1 U) ; to be FIG . 1
I+ Il CH3-S-0-CHZ-CHZ-C-N, G 0 Pipoaulfan
~
II II N-C-CHZ-OS-CH3
(Ancyte)
CH3-S-0-CHZ-CHZ-CH Z-CHZ-O-S-CH3
li
II
0
0 Busulfan (Myleran®)
0 . ._ 0 u , Br-CHZ-CHZ-C-N, , ~N-C-CHZ-CHZ-Br Pipobruman (Vercyte ®) incorporated into DNA (11) ; to react with cyateine and other thiola in vivo (la) ; t~~ ini~ibit .)NA synthesis (13) ; to inhi~it mitosis and cause giant cell '.urination (14) ; to inhibit spermatogenesis in rata (15) ; to cause mutations in ~?rosophilia
(16) ; and to inhibit crystalline trioeephosphate dehydrogenase (17) .
Uf course we could expect a similar multiplicity of action with pipoaulfan and consequently difficulty in determining the mechanism of ite antitumor activity .
Nevertheless, it was of interest to question how it might be typical
or atypical of this class of drugs . Materials and Methods î,e11 Line Ehrlich ascitea carcinoma subline ES-III a'/357 was obtained from A.D . Little ~ Co ., Cambridge, Mass ., and maintained here by weekly transplant in
Effect od Alkylating Agents
Vol . 10, No . 11
807
Swiss-Webster male mice (25-35 gm) . Cell Couat and Viability Cell coasts ware done with the aid of a hemocytometer .
Viability was
calculated from the total number of cells minus the number of cells staining with Trypaa Blue (0 .07x) divided by the total number of cells . Assay of Macramolecular Synthesis At appropriate times, cell were removed from the peritoneal cavity and washed in Eagle's M~I, pH 7 .
Five to ten million cells from each animal were
suspended, in duplicate, in two mls of Eagle's MEM (Hank's salts) with 2mM glutamine, pH 7 .2, containing radioactive precursor and incubated for 1 hour at 37 ° C in air, with shaking .
Protein, RNA and DNA synthesis was measured
as TCA insoluble radioactivity from uridine (500 mCi/mmol) sad
14 C - leucine (311 mCi/mmol),
3H -
3fi -thymidine (500 mCi/mmol) respectively .
The
reaction was stopped with 3 ml of Sx TCA containing 1 mg/ml cold precursor and the mixture centrifuged .
The cells were washed again is TCA, then in
ethanol and twice more is athaaol :ether (3 :1) .
The residue was dried in a
water bath, dissolved in 0 .5 ml formic acid and the radioactivity determined in liquid scintillation spectrometer . Results and Discussion Table 1 gives the results of a representative experiment designed to teat the effects of piposulfaa on DNA, RNA and protein synthesis is vivo .
Seven
days after transplaatatioa (106 cells), six mice were randomized into two equal groups .
Group C received two iatraperitoneal (i .p .) injections of 0 .5
ml saline and group X vas given two injections of 100 mg/kg of pipoaulfan i .p . in saline seven hours apart .
The aaimala were sacrificed 24 hours after the
first treatment sad the tumor cells processed as described under Materials sad Methods .
The data show a 48S increase in the rata of protein synthesis,
no significant change in. RNA synthesis and a 25x decrease in the rate of DNA synthesis in carcinoma cells from animals treated with piposulfan .
Evidently
these effects would precede a "clinical" response to pipoaulfan since neither
808
Effect oaf Alkylating Agents
Yol . 10, No. 11
cell viability nor cell count was significantly altered bq the drug at this time .
Routine microscopic examination showed no noticeable difference in
appearance between control and experimental calls . A similar pattern of activity was found in preliminary experiments using the secitea form of the 4lalker 256 sarcoma is rate . TABLE 1 Effects of Piposulfan on Macromolecular Synthesis and Cell Count in Ehrlich Asc hes Carcinoma After One Day . of Treatment Total Cell Count Lilliona~
Cell Viability
Mouse
_TCA Insol . Radioactivity (CPM) 1 Leuçine Uridine Th~m idine
C1
17,243
82,289
22,849
225
98
C2
14,722
80,736
22,068
400
90
C3
14,485
88,641
17,940
190
95
Average 2 15,480
83,922
20,952
272
94
± 879
*- 2407
~ 1523
X1
27,508
83,765
17,488
189
95
X2
23,785
80,014
17,645
204
95
X3
17,885
94,557
15,792
330
90
Average
23,059
86,109
15,792
241
93
t 2802
± 4360
± 1775
148
102
75
89
99
~ of Control
7
l Cell number was identical in all tubes,. 2 Averages are given ± S .E . Table 2 shows the results of an experiment similar to the one above except that the tumors were treated for two days (2 x 100 mg/kg/day) . Here we observe pronounced effects on protein and DNA synthesis again without
Vol . 10, No. 11
Effect ad Alkylating Agents
significant change in ßNA synthesis .
609
Dote also the marked reduction in cell
count indicating a therapeutic response to piposulfan .
Since viability re-
mains unchanged at this time, ae might surmise that mitosis has been arrested by the drug . cell volume
Consistent with this, ae observed a definite lncreaae in the (giant cells) of the carcinoma cells from treated animals after
tao days . TABLE 2 ßffecte of Piposulfan on Macromolecular 5yntheaia and Cell Count in Ehrlich Asches Carcinoma After ~o Days of Treatment Total Cell Count (millions)
Cell Viability X
22,537
189
95
95,527
21,232
180
86
19,587
72,437
24,599
105
98
Average2 19,357
86,984
22,786
158
93
t 373
t 7311
t 980
X1
29,.915
66,522
13,111
39
100
XZ
53,983
95,456
17,012
60
94
X3
40,177
76,924
15,118
39
93
Average
41,358
79,634
15,080
46
93
t 6973
t 8462
± 1126
213
91
66
29
103
Mouse
TCA Insol . ßadioactivity (CPM)1 Laudas Uridino Thymidine
C1
19,857
92,988
C2
18,628
C3
X of Control
1 Cell number ass identical in all tubes. 2 Averages are given t S .ß . Table 3 sussarises additional experiments similar to the ones described above .
It may be seen that the effects of piposulfaa on protein sad DdiA syn-
thesis became evident betaean 16 and 24 hours after drug administration and
810
Effect ad Alkylating Agents
becona pronauad at later times .
Vol. 10, No . 11
We do not know if the drug affects the syn-
thasis of DNA sad protein independently of each other .
It would be interesting
to learn what effect piposulfan may have on protein synthesis in systems where nucleic acid synthesis is not present, as in reticulocytes, for example . TABLE 3 Effects of Pipoaulfan on Labeling of DNA and Protein
Bty. #1 1
(3)
2
(6)
3
Treatmant2 (nft/ka i .p .)
Time of Sacrifica3 (Hours)
504
Incorporation of Precursor as of Control ThS+midine Leucine
x
16
90
114
2 x 25
24
89
142
(6)
2 x 50
24
77
149
4
(3)
50
40
73
196
5
(3)
2 x 50
48
56
296
1 The number of mice per group is given in parentheses . 2 Where two injections were given they were seven hours apart on the first day of treatment, 3 Time given from the first injection. 4 The i .p . LD50 of piposulfan in Swiss-Webster mice is 400 mg/kg (4) . Table 4 gives the results of experiments in which DNA synthesis was measured 2 and 4 hours after a single intraperitoneal dose of 200 mg/kg of pipoaulfan or pipobroman (3 animals/group) .
Pipobraman, which seems to act
like a "typical" alkylating agent (see below), differs from piposulfan in having bramiaa instead of methylsulfonic acid as the leaving group in reactions with nuclaophilas (see Figure 1) and it is interesting to note that this makes quite a difference biologically .
In contrast to drastic inhibition of DNA
synthesis by pipobrae~an, piposulfaa had no effect at 2 and 4 hours after drug adniaistration .
Neither drug affected protein synthesis at these times but
pipobranan slightly inhibited (ca, 25x) RNA synthesis at 4 hours.
Vol. 10, No . 11
Effect ad Alkylating Agents
611
TABLE 4 Comparison of the Effect of Piposulfaa sad Pipobroman on DNA Syathesis l Two sad Four Hours After Drug Administration
Sam~e2
Time of Sacrifice
Average Radioactivity t S~E~ ~PM)
Relative Açtivity
Controle
2
12805 t 867
100
Piposulfan
2
13809 t 451
108
Pipobroman
2
2629 t 360
21
Controls
4
8773 t 1136
100
Piposulfan
4
9301 t 487
106
Pipobroman
4
1770 t 367
20
1 Incorporation of 3H-thymidine into TCA insoluble material . 2 Each group had three mice . 3 Cell number Was identical in all tubes . Among other possibilities, such se inhibition of the biosynthesis of a vital cell component and its subsequent exhaustion from the cell, the delayed onset of activity with pipoaulfan may indicate a site of action which ie available during only part of the cell cycle .
Future experiments using
synchronized cultures could prove useful to our understanding of its mode of action and of cell biology .
Its effect on protein syatheeis merits further
study in itself and here also pipoaulfan may prove to be a useful biochemical tool . References 1.
J . A . CARBON, S . M . BREM aàd J . D, RATAJCZYK, Abstr . Paper, 139th Meeting, Am . Chem . Soc . (1961) .
2.
R, J . STEIN, J . A . CARBON, J . LANGDON and R, K, RICHARDS, J . Lab . Clin . Med . 5 6 :949 (1960) .
3.
Sloan-KetterinA Tumor Spectrum Studies , SK#24591, (1961) .
4.
T . J, MCNAIR, F . A, WIBIN, E . T, HOPPE, J, L . SCHMIDT and F, A, dePEYSTER, J . S4rft . Res . _, 130 (1963) .
612
Effect of Alkylating Agents
Vol. 10, No . 11
5.
N. A, NELSON, R, W. TALLEY, M. L. REED, A, M, EVANS, B. L . ISAACS, P. RUFFItAN and J. LOUIS, Clin . Pharmacol, Therau . 8, 385 (1967) .
6.
A. HADDOW ;
7.
G, P, WHEELER,
Cancer Rea .
22, 651 (1962) .
8,
G, P, WARWICK,
Cancer Rea,
23, 1315 (1963) .
9.
H. VERMIAZD and F . F, COLL7N,
10 .
E, G. TRAMS, M, V . NADRARNI, V. DEQUATTRO, D, MAStiGWYN-DAVIES and P, K. SMITH, Biochem, Pharmacol. 2, 7 (1959) .
Physiopatholomr of Cancer , p . 602, N, Y., Hoeber Prese (1959) .
Cancer
21, 58 (1968) .
11 .
E, G. TRAMS, M. V, NADKARNI and P. K. SMITH,
12 .
J . J. ROBERTS and G. P. WARWICK, Biochem. Pha:macol .
13 .
J. G. LI, M. E, LEONARD and G . HARRISON,
14 .
R, BASSLEER,
15 .
K. A. AHLQUIST,
16 .
0, G. FAHM1f and J, J. FAHMY,
17 .
H. HOLZER,
Comet . Read . Soc . Biol . J. Reurod . Fart .
Cancer Rea.
Cancer
21, 560 (1961) .
6, 216 (1961 ; .
9, 963 (1956) .
~, 237 (1965) .
12, 377 (1966) .
J. Genetics
46, 361 (1961) .
Die Mediziniache , 576 (1956) ; CA 50, 10257 (1966) .