Vol.
176,
No.
1, 1991
April
15,
1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 505-510
EFFECTS OF A FECAPENTAENE ON PROTEIN KINASE C
Sadayori
Hoshina,' I.
Marius Ueffing, Masami Bernard Weinstein'
Comprehensive Cancer Center Columbia University,
Received
March
and Institute New York,
Morotomi,
of Cancer NY 10032
and
Research
6, 1991
Protein kinase C (PKC) is a Ca"+- and phospholipid-dependent serine and threonine protein kinase which binds and is activated by tumor promoters such as the ohorbol ester 12-O-tetradecanovlphorbol-13-acetate (TPA). PKC b vitro by phosphatidylserine (PS) plus -Ca". We can be actibated report here that the compound fecapentaene-12 can replace the requirement for PS in the activation of PKC by Ca". In addition, at low concentrations fecapentaene-12 can enhance the activation of PKC by It can also either enhance or inhibit activation of PKC Ca2+ and PS. depending on the assay conditions. by the tumor promoter teleocidin, These results are of interest since fecapentaene is known to be a potent mutagen that is ,produced by Bacteroides species present in the lumen of the human The present studies raise the possibility that this compound might colon. also play a role in colon cancer by altering the activity .of PKC. 0 1991 Academic Press, Inc.
There is now considerable experimental and epidemiological evidence that the human colonic mucosa is frequently exposed to fecapentaenes, a group of potent mutagens produced by certain Bacteroides from polyunsaturated ether Fecapentaenes exhibit a specific W-triplet absorbance phospholipids (1). spectrum, are highly unstable, and undergo degradation when exposed to light, oxygen, or acidic pH (2). a synthetic prototype for the fecapentaenes, is highly Fecapentaene-12, genotoxic to human cells since it induces DNA-single strand breaks, chromosomal unscheduled DNA synthesis, aberrations, sister chromatid and specific mutations (3,4,5). In vitro studies indicate that exchanges,, both DNA-single strand breaks and alkali-labile fecapentaenes can cause and lead to hydroxylation of the C-8 position of guanine residues in sites,
' Present Minato-ku,
Address: Jikei University Tokyo 105, JAPAN.
2 To whom correspondence Abbreviations: phosphatidylserine; growth factor;
should
School
of Hedicine,3-25-8
Nishi-shinbashi,
be addressed.
PKC, protein kinase C; FP, TPA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, phenylmethylsulfonyl fluoride.
fecapentaene; EGF,
PS, epidermal
0006-291X/91 505
Ail
Copyright 0 1991 rights of reproduction
$1.50
by Academic Press. Inc. in my form reserved.
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
fecapentaene-12 reacts directly with glutathione and Furthermore, DNA (6). can cause thiol depletion (7). Protein kinase C (PKC) is a Ga'+ and phospholipid-dependent protein kfnase which binds and is activated by certain tumor promoters, such as the The activation of PKC appears to be a critical phorbol ester TPA (8,9). event in tumor promotion. We report here that fecapentaene-12 can substitute for phospholipid as a cofactor in the activation of PKC. These results, contain a glycerol moiety in taken together with the fact that fecapentaenes their molecular structure, may be of interest with respect to a possible role of fecapentaenes as promoters of colon carcinogenesis. MATERIALS
AND WETHODS
Materials
Methods nmol~~~~m~~~mgpU~~fi~rdev~~~~l~a~e~~~~~dto(l~~ spec~nf~~meacat~tvi?~tyofw~32 stimulated About 10 fold by 800 PM Ca +' plus 100 pM PS. PKC were performed as previously described (15). The standard reaction toethanol, 1OmM ~~~~~",es contained 20mM Tris HCl at H 7 2, 5mM 2-merca 800 pM CaCl+, ;@$ puergiefnidesd *~~~~~~~t:~~~~~~~l~ac~~~~o~e~'pmol' ic modifications are desciibed and 3in 'g the initiated by the and incubated for a period of ten minutes at 3O"C, ad ition of the enzyme, which is in the linear phase of the time course. Reactions were terminated and the radioactivit incor orated into the EGF on phosphocellulose paper, receptor peptide was measured as previously descr f bed (14 5 . RESULTS Our standard assay for PKC contains 100 fl phosphatidylserine (PS) and 800 fl Ca2+, since these concentrations of the respective optimal stimulation of enzyme activity (12). components provide Since various lipids including certain fatty acids can substitute for PS as the lipid cofactor (9,12), we examined whether this was the case with fecapentaene-12 (FP). Figure 1 indicates that FP produced a concentration-dependent enhancement of PKG activity when substituted for PS assayed in the presence of 800 fl Ca'+. and Significant stimulation was obtained with as little as 4 fl FP, and the response was linear up to 160 $4. With the latter concentration of FP, PKC activity was 121 pmole 32P/min, which was approximately equal to that obtained Thus FP is about 60% as potent as PS as a lipid cofactor with 100 pM PS. for PKG activation. We next examined the Ca2+ optimum for FP activation of PKC. Figure 2 indicates that, as with PS (8), the optimum Ca2+ concentration was Figure 3 indicates that FP produced a biphasic effect on about 800 PM.
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
80
Figure
1.
The effect ;ypity.
and
additional
of increasing PKC activity
the
concentrations was assayed
indicated
details
see
&.&l&l
and
160
of in
concentrations
COMMUNICATIONS
the
of
Hethods.
fecapentaene-12 resence of P ecapentaene.
on PKC 800Fo;M
assays that also contained 100 $4 PS, when assayed in the presence of At a low 800 pl4 Ca2+. concentration (4 PM) FP produced a enhancement (about 1.7 fold) of enzyme activity. further This effect diminished with higher concentrations of FP (lo-4Ofl), and higher concentrations of FP (80 - 160 PM) partially inhibited the activity obtained with PS alone. The latter effect may simply reflect the fact that even high concentrations of PS can inhibit PKC activity (unpublished data). PKC
Previous studies indicate that the tumor promoters TPA and teleocidin are potent activators of PKC in the presence of PS (8,15). This effect is maximal at very low concentrations of Ca2+ (10 $4), and can be seen even in the absence of added Ca2+ and the presence of EGTA (15). Thus, agents markedly diminish (and can even abolish) the Ca2+ these
70-
350
-
c 60‘E 502
300
-
2
.-c g250-
400a, 30-
PI ~
E 20-
-z
a
E Q
IO-
02
0
800
pM Figure
2. Effect
@ia4f Figure
3.
IOO50-
03
I
[Ca*‘l100
IO
ISO-
on In this FP gave
For additional same experiment a PKC activity
0
I 04
I IO
I 20
80
F PI, y: fecapentaene-stimulated PKC in the presence of 80 $4 concentrations of Ca" details see Uateriai ir% the addition 100 @f ps of 132 pmol ' P/min/mg at
resence p+f effect of fecapentaene-12 on PKC activity in the Assays were performed in the presence of 8 8 0 $4 Ca anh 100 JIM PS and the indicated concentraii;ans of fecapentaene-12. For additional details see J4at r 1 and Methods.
E
507
I 160
Vol.
176,
No.
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
aJ 0 E a
40
0
0
8
40
iF P;p,,
5
Figure
4.
Stimulationooff x:9:' Methods.
Figure
5. Inhibition T;vyed
of
i;
PKC activity teleocidin PS. For
b (280 additional
fecapentaene-12 nM) and details
ts
fecapentaery;-12.
of PKC activity tazd
addit Ponal details '
pt;le,sence indicated
see Material
10
$4 Ca concentrhtions
and Methods.
10
(8-40,+fl)inint;i fl Ca see Hat'erial
200
PKC activit nM teleoc of FP.
anQ was Pdin, For
requirement of the enzyme. We found that teleocfdin (200 nM) was also a low concentrations of FP (S-20 potent activator of PKC when relatively PM) were substituted for PS, in assays containing a low concentration of Higher concentrations Ca'+ (10 fl) (Fig.4). of FP did not further In the presence of 100 fl PS, however, the addition enhance this effect. of FP did not further enhance the high activity obtained with teleocidin (200 Indeed, nM) and 10 PM Ca" (Figure 5). under these conditions, FP (S-40 fi) had an inhibitory effect on PKC activity (Figure 5). During the course of these studies we observed that although a lipid cofactor, either PS or FP, was required for maximal stimulation of PKC, significant and reproducible stimulation of enzyme activity by teleocidin was seen in the absence of PS or FP. This result is apparent in Figure 4. In this experiment, the value obtained with 200 nM teleocidin in the absence of an added lipid cofactor was about 7.3 times the blank value (i.e., minus teleocidin, minus PS and FP, plus 10 fi Ca'+). We have also observed significant stimulation of enzyme activity by teleocidin in the absence of a lipid cofactor using a purified preparation of the B1 isoform of PKC (14) (data not shown here). Other investigators have recently observed that TPA can also cause significant stimulation of PKC activity in the absence of a lipid cofactor, although the value obtained was only about 20% of that obtained in the presence of PS (10).
DISCUSSION
Although PKC was originally defined as a Ca'+and phospholipiddependent enzyme (S), it is now apparent that a number of factors can influence its in vitro activity. The addition of certain diacylglycerols or the tumor promoters TPA, teleocidin or aplysiatoxin activates the enzyme and the Ca2+ requirement markedly reduces (9,12,15). The nature of the lipid cofactor has also been investigated in detail. In general, anionic lipids are active, including the phospholipids PS and PI (11) as well as 508
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
polyphosphorylated phosphatidylinositols (12). Certain fatty acids can also substitute for the phospholipid cofactor, including arachidonic acid (9), and retinoic acid (13), and also certain hydroperoxy-derivatives of unsaturated fatty acids (15). The present study indicates that the compound fecapentaene-12 (FC), a 1-(1-glycero) dodeca-1,3,5,7,9-pentaene, can also serve as the lipid cofactor for rat brain PKC with a potency that is about 60-70% that of PS (Figure l), depending on the Ca2+ concentration. PS is the most active of the known lipid cofactors. As with PS, the optimum Ca2+ concentration in assays containing FC is about 800 @+!, and in assays containing the potent PKC activator teleocidin the Ca2+ requirement is markedly reduced (Figures 2-4). The preparation of rat brain PKC used in the present studies is probably a mixture of at least three isoforms of PKC, SO it is difficult to know which of these isoforms is stimulated by FC. Since we obtained high enzyme activity with FC we assume that the stimulation is not confined to a minor isoform. We have also similar results with FC using purified PKC observed isolated from 81 crells developed in our laboratory (14) that overexpress this specific isoform (unpublished data). The compound FC was first identified as a potent bacterial mutagen in human feces, but its relevance to the causation of human colon cancer is not clear at the present time (1,7). Because of its mutagenic activity one might assume from previous studies that if it does play a role it would be through its genotoxic effects, and, therefore, might act at the stage of initiation and/or progression. The present results raise the possibility that it could also act as a tumor promoter by enhancing PKC activity in the colonic epithelium. Since the Bacteroides that produce fecapentaenes are usually in direct contact with the colon epithelium the latter cells might be exposed to appreciable concentrations of fecapentaenes. We should emphasize, however, that the present results were obtained with pmolar concentrations of FC, In and we do not know if these levels are attained under in viva conditions. addition, it is not known whether Jn vivo the lipid cofactor for PKC is rate limiting. Regardless of their biologic significance, the present studies expand our information on the range of lipid structures than can serve as cofactors of FP has an unusual chemical structure since it has an aliphatic chain of PKC. five double-bonded carbon atoms terminated at one end by a diglyceride-like Thus, it shares properties with certain other lipid cofactors, as structure. The incidental finding that the tumor well as the DAG activators of PKC. promoter teleocidin can cause significant activation of PKC even in the absence of a lipid cofactor (Figure 4) may be of interest in terms of understanding mechanisms of PKC activation at the molecular level. ACKNOWLEDGMENTS awards from the Yakult-Honsha Company This research was supported b and the Aaron Diamond Foun Bation (New York City) to I.B.W. Sheila (Tokyo H. Wash ington provided valuable assistance in preparation of this manuscript.
REFERENCES 1. Van Tassel1 (1989) Lipids
R.L., Piccariello, 24, 454-459.
T., 509
Kingston,
D.G.K.,
and Wilkins,
T.D.
Vol.
176,
No.
1, 1991
BIOCHEMICAL
Suz ki K., 52l?-5513.
AND
3.
Bauchinger, Mutation Resea:ch
4.
Venitt,
5.
Shino ama M T. (1);89)'Mutation
6.
Hinzman, M.J., Novotn p, C., Carcinogenesis 8, 1475- 479.
7.
Dypbukt, Harris 6058-6063.
J.M., C.C.
Edman, C.C., Sungqvist, K., Kakefuda, T., and Grafstrom, R.C. (1989) Cancer
8.
$$;;leo,
J.P.
and
9.
Nishizuka,
Y.
(1988)
10.
Couturier, A Res. Commun.
11.
Berridge,
12.
O'Brian 214, 339-3&A"
13.
H.J.
:f?l&P.i57-161.
S&mid,
&?9r,th289'-3f:'
and Bosworth,
D. (1988)
Wakabayashi Research
25i:
Nature
?fun;;hita, - .
Arthur, Emeler,
W.L., C.A.,
Pfaendler,
P.
(1984)
H.R.
(1988)
3, 169-173. K.,
Nagao,
and
Shamsuddin,
(1985)
H.,
and
Sugimura,
A.M.
(1987)
Plummer, Research
Carcinogenesis
Castagna, (1989) and
Butcher,
G.M., Johnpon M.D., Klrschmeier, ., Weinstein,
A.
and Yate,
6,
S.M., 49,
213-217,
661-665.
and R.F.
and
Mutagenesis
I.B. 334,
J.J.,
E.
Ullah,
Weinstein,
and Irvine,
Krepinsky,
COMMUNICATIONS
Gu uta Sc!enck
S.,
R.,
RESEARCH
2.
iij,
Bruce,
RIOPHYSICAL
M.
Nature
(1988) 341,
Weinstein, F.R.,
and
Fontana,
zey,. .,
15.
O'Brian C.A. Arcoleo J.P House G.M. and in: Cancer Cells: GrowGh Fac
176,
No.
1, 1991
April
15,
1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 505-510
EFFECTS OF A FECAPENTAENE ON PROTEIN KINASE C
Sadayori
Hoshina,' I.
Marius Ueffing, Masami Bernard Weinstein'
Comprehensive Cancer Center Columbia University,
Received
March
and Institute New York,
Morotomi,
of Cancer NY 10032
and
Research
6, 1991
Protein kinase C (PKC) is a Ca"+- and phospholipid-dependent serine and threonine protein kinase which binds and is activated by tumor promoters such as the ohorbol ester 12-O-tetradecanovlphorbol-13-acetate (TPA). PKC b vitro by phosphatidylserine (PS) plus -Ca". We can be actibated report here that the compound fecapentaene-12 can replace the requirement for PS in the activation of PKC by Ca". In addition, at low concentrations fecapentaene-12 can enhance the activation of PKC by It can also either enhance or inhibit activation of PKC Ca2+ and PS. depending on the assay conditions. by the tumor promoter teleocidin, These results are of interest since fecapentaene is known to be a potent mutagen that is ,produced by Bacteroides species present in the lumen of the human The present studies raise the possibility that this compound might colon. also play a role in colon cancer by altering the activity .of PKC. 0 1991 Academic Press, Inc.
There is now considerable experimental and epidemiological evidence that the human colonic mucosa is frequently exposed to fecapentaenes, a group of potent mutagens produced by certain Bacteroides from polyunsaturated ether Fecapentaenes exhibit a specific W-triplet absorbance phospholipids (1). spectrum, are highly unstable, and undergo degradation when exposed to light, oxygen, or acidic pH (2). a synthetic prototype for the fecapentaenes, is highly Fecapentaene-12, genotoxic to human cells since it induces DNA-single strand breaks, chromosomal unscheduled DNA synthesis, aberrations, sister chromatid and specific mutations (3,4,5). In vitro studies indicate that exchanges,, both DNA-single strand breaks and alkali-labile fecapentaenes can cause and lead to hydroxylation of the C-8 position of guanine residues in sites,
' Present Minato-ku,
Address: Jikei University Tokyo 105, JAPAN.
2 To whom correspondence Abbreviations: phosphatidylserine; growth factor;
should
School
of Hedicine,3-25-8
Nishi-shinbashi,
be addressed.
PKC, protein kinase C; FP, TPA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, phenylmethylsulfonyl fluoride.
fecapentaene; EGF,
PS, epidermal
0006-291X/91 505
Ail
Copyright 0 1991 rights of reproduction
$1.50
by Academic Press. Inc. in my form reserved.
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
fecapentaene-12 reacts directly with glutathione and Furthermore, DNA (6). can cause thiol depletion (7). Protein kinase C (PKC) is a Ga'+ and phospholipid-dependent protein kfnase which binds and is activated by certain tumor promoters, such as the The activation of PKC appears to be a critical phorbol ester TPA (8,9). event in tumor promotion. We report here that fecapentaene-12 can substitute for phospholipid as a cofactor in the activation of PKC. These results, contain a glycerol moiety in taken together with the fact that fecapentaenes their molecular structure, may be of interest with respect to a possible role of fecapentaenes as promoters of colon carcinogenesis. MATERIALS
AND WETHODS
Materials
Methods nmol~~~~m~~~mgpU~~fi~rdev~~~~l~a~e~~~~~dto(l~~ spec~nf~~meacat~tvi?~tyofw~32 stimulated About 10 fold by 800 PM Ca +' plus 100 pM PS. PKC were performed as previously described (15). The standard reaction toethanol, 1OmM ~~~~~",es contained 20mM Tris HCl at H 7 2, 5mM 2-merca 800 pM CaCl+, ;@$ puergiefnidesd *~~~~~~~t:~~~~~~~l~ac~~~~o~e~'pmol' ic modifications are desciibed and 3in 'g the initiated by the and incubated for a period of ten minutes at 3O"C, ad ition of the enzyme, which is in the linear phase of the time course. Reactions were terminated and the radioactivit incor orated into the EGF on phosphocellulose paper, receptor peptide was measured as previously descr f bed (14 5 . RESULTS Our standard assay for PKC contains 100 fl phosphatidylserine (PS) and 800 fl Ca2+, since these concentrations of the respective optimal stimulation of enzyme activity (12). components provide Since various lipids including certain fatty acids can substitute for PS as the lipid cofactor (9,12), we examined whether this was the case with fecapentaene-12 (FP). Figure 1 indicates that FP produced a concentration-dependent enhancement of PKG activity when substituted for PS assayed in the presence of 800 fl Ca'+. and Significant stimulation was obtained with as little as 4 fl FP, and the response was linear up to 160 $4. With the latter concentration of FP, PKC activity was 121 pmole 32P/min, which was approximately equal to that obtained Thus FP is about 60% as potent as PS as a lipid cofactor with 100 pM PS. for PKG activation. We next examined the Ca2+ optimum for FP activation of PKC. Figure 2 indicates that, as with PS (8), the optimum Ca2+ concentration was Figure 3 indicates that FP produced a biphasic effect on about 800 PM.
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
80
Figure
1.
The effect ;ypity.
and
additional
of increasing PKC activity
the
concentrations was assayed
indicated
details
see
&.&l&l
and
160
of in
concentrations
COMMUNICATIONS
the
of
Hethods.
fecapentaene-12 resence of P ecapentaene.
on PKC 800Fo;M
assays that also contained 100 $4 PS, when assayed in the presence of At a low 800 pl4 Ca2+. concentration (4 PM) FP produced a enhancement (about 1.7 fold) of enzyme activity. further This effect diminished with higher concentrations of FP (lo-4Ofl), and higher concentrations of FP (80 - 160 PM) partially inhibited the activity obtained with PS alone. The latter effect may simply reflect the fact that even high concentrations of PS can inhibit PKC activity (unpublished data). PKC
Previous studies indicate that the tumor promoters TPA and teleocidin are potent activators of PKC in the presence of PS (8,15). This effect is maximal at very low concentrations of Ca2+ (10 $4), and can be seen even in the absence of added Ca2+ and the presence of EGTA (15). Thus, agents markedly diminish (and can even abolish) the Ca2+ these
70-
350
-
c 60‘E 502
300
-
2
.-c g250-
400a, 30-
PI ~
E 20-
-z
a
E Q
IO-
02
0
800
pM Figure
2. Effect
@ia4f Figure
3.
IOO50-
03
I
[Ca*‘l100
IO
ISO-
on In this FP gave
For additional same experiment a PKC activity
0
I 04
I IO
I 20
80
F PI, y: fecapentaene-stimulated PKC in the presence of 80 $4 concentrations of Ca" details see Uateriai ir% the addition 100 @f ps of 132 pmol ' P/min/mg at
resence p+f effect of fecapentaene-12 on PKC activity in the Assays were performed in the presence of 8 8 0 $4 Ca anh 100 JIM PS and the indicated concentraii;ans of fecapentaene-12. For additional details see J4at r 1 and Methods.
E
507
I 160
Vol.
176,
No.
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
aJ 0 E a
40
0
0
8
40
iF P;p,,
5
Figure
4.
Stimulationooff x:9:' Methods.
Figure
5. Inhibition T;vyed
of
i;
PKC activity teleocidin PS. For
b (280 additional
fecapentaene-12 nM) and details
ts
fecapentaery;-12.
of PKC activity tazd
addit Ponal details '
pt;le,sence indicated
see Material
10
$4 Ca concentrhtions
and Methods.
10
(8-40,+fl)inint;i fl Ca see Hat'erial
200
PKC activit nM teleoc of FP.
anQ was Pdin, For
requirement of the enzyme. We found that teleocfdin (200 nM) was also a low concentrations of FP (S-20 potent activator of PKC when relatively PM) were substituted for PS, in assays containing a low concentration of Higher concentrations Ca'+ (10 fl) (Fig.4). of FP did not further In the presence of 100 fl PS, however, the addition enhance this effect. of FP did not further enhance the high activity obtained with teleocidin (200 Indeed, nM) and 10 PM Ca" (Figure 5). under these conditions, FP (S-40 fi) had an inhibitory effect on PKC activity (Figure 5). During the course of these studies we observed that although a lipid cofactor, either PS or FP, was required for maximal stimulation of PKC, significant and reproducible stimulation of enzyme activity by teleocidin was seen in the absence of PS or FP. This result is apparent in Figure 4. In this experiment, the value obtained with 200 nM teleocidin in the absence of an added lipid cofactor was about 7.3 times the blank value (i.e., minus teleocidin, minus PS and FP, plus 10 fi Ca'+). We have also observed significant stimulation of enzyme activity by teleocidin in the absence of a lipid cofactor using a purified preparation of the B1 isoform of PKC (14) (data not shown here). Other investigators have recently observed that TPA can also cause significant stimulation of PKC activity in the absence of a lipid cofactor, although the value obtained was only about 20% of that obtained in the presence of PS (10).
DISCUSSION
Although PKC was originally defined as a Ca'+and phospholipiddependent enzyme (S), it is now apparent that a number of factors can influence its in vitro activity. The addition of certain diacylglycerols or the tumor promoters TPA, teleocidin or aplysiatoxin activates the enzyme and the Ca2+ requirement markedly reduces (9,12,15). The nature of the lipid cofactor has also been investigated in detail. In general, anionic lipids are active, including the phospholipids PS and PI (11) as well as 508
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
polyphosphorylated phosphatidylinositols (12). Certain fatty acids can also substitute for the phospholipid cofactor, including arachidonic acid (9), and retinoic acid (13), and also certain hydroperoxy-derivatives of unsaturated fatty acids (15). The present study indicates that the compound fecapentaene-12 (FC), a 1-(1-glycero) dodeca-1,3,5,7,9-pentaene, can also serve as the lipid cofactor for rat brain PKC with a potency that is about 60-70% that of PS (Figure l), depending on the Ca2+ concentration. PS is the most active of the known lipid cofactors. As with PS, the optimum Ca2+ concentration in assays containing FC is about 800 @+!, and in assays containing the potent PKC activator teleocidin the Ca2+ requirement is markedly reduced (Figures 2-4). The preparation of rat brain PKC used in the present studies is probably a mixture of at least three isoforms of PKC, SO it is difficult to know which of these isoforms is stimulated by FC. Since we obtained high enzyme activity with FC we assume that the stimulation is not confined to a minor isoform. We have also similar results with FC using purified PKC observed isolated from 81 crells developed in our laboratory (14) that overexpress this specific isoform (unpublished data). The compound FC was first identified as a potent bacterial mutagen in human feces, but its relevance to the causation of human colon cancer is not clear at the present time (1,7). Because of its mutagenic activity one might assume from previous studies that if it does play a role it would be through its genotoxic effects, and, therefore, might act at the stage of initiation and/or progression. The present results raise the possibility that it could also act as a tumor promoter by enhancing PKC activity in the colonic epithelium. Since the Bacteroides that produce fecapentaenes are usually in direct contact with the colon epithelium the latter cells might be exposed to appreciable concentrations of fecapentaenes. We should emphasize, however, that the present results were obtained with pmolar concentrations of FC, In and we do not know if these levels are attained under in viva conditions. addition, it is not known whether Jn vivo the lipid cofactor for PKC is rate limiting. Regardless of their biologic significance, the present studies expand our information on the range of lipid structures than can serve as cofactors of FP has an unusual chemical structure since it has an aliphatic chain of PKC. five double-bonded carbon atoms terminated at one end by a diglyceride-like Thus, it shares properties with certain other lipid cofactors, as structure. The incidental finding that the tumor well as the DAG activators of PKC. promoter teleocidin can cause significant activation of PKC even in the absence of a lipid cofactor (Figure 4) may be of interest in terms of understanding mechanisms of PKC activation at the molecular level. ACKNOWLEDGMENTS awards from the Yakult-Honsha Company This research was supported b and the Aaron Diamond Foun Bation (New York City) to I.B.W. Sheila (Tokyo H. Wash ington provided valuable assistance in preparation of this manuscript.
REFERENCES 1. Van Tassel1 (1989) Lipids
R.L., Piccariello, 24, 454-459.
T., 509
Kingston,
D.G.K.,
and Wilkins,
T.D.
Vol.
176,
No.
1, 1991
BIOCHEMICAL
Suz ki K., 52l?-5513.
AND
3.
Bauchinger, Mutation Resea:ch
4.
Venitt,
5.
Shino ama M T. (1);89)'Mutation
6.
Hinzman, M.J., Novotn p, C., Carcinogenesis 8, 1475- 479.
7.
Dypbukt, Harris 6058-6063.
J.M., C.C.
Edman, C.C., Sungqvist, K., Kakefuda, T., and Grafstrom, R.C. (1989) Cancer
8.
$$;;leo,
J.P.
and
9.
Nishizuka,
Y.
(1988)
10.
Couturier, A Res. Commun.
11.
Berridge,
12.
O'Brian 214, 339-3&A"
13.
H.J.
:f?l&P.i57-161.
S&mid,
&?9r,th289'-3f:'
and Bosworth,
D. (1988)
Wakabayashi Research
25i:
Nature
?fun;;hita, - .
Arthur, Emeler,
W.L., C.A.,
Pfaendler,
P.
(1984)
H.R.
(1988)
3, 169-173. K.,
Nagao,
and
Shamsuddin,
(1985)
H.,
and
Sugimura,
A.M.
(1987)
Plummer, Research
Carcinogenesis
Castagna, (1989) and
Butcher,
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