Transplasma membrane redox stimulates HeLa cell growth

Vol. 125, No. 2, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1984 Pages 649-654

Transplasma Membrane Redox Stimulates HeLa Cell Growth

I. L. Sun, F. L. Crane

Department of Biological Sciences Purdue IJniversity

West Lafayette, IN 47907

H. Liiw and C. Grebing

Endocrinology Department Karolinska Institute

Stockholm, Sweden

Received November 1, 1984

Impermeable ferricyanide stimulates the growth of HeLa cells in absence of fetal bovine serum or other growth factors. A series of impermeable oxidants with redox potentials down to -125 mV stimulate equivalent growth. All of these oxidants are reduced by the transplasma membrane electron transport system. Oxidants with redox potentials below -175 mV are not reduced by the transmembrane electron transport and do not stimulate growth. Insulin which stimulates growth in absence of serum also stimulates transmembrane ferricyanide reduction. Ferricyanide increases growth in presence of insulin. Antitumor drugs, which inhibit HeLa cell growth, inhibit the transplasma membrane redox system. Transplasma membrane electron transport is accompanied by proton release from HeLa cells. B 1984

Academic Press, Inc.

Stimulation of the growth of melanoma cells by external ferricyanide

has been demonstrated by Ellem and Kay (1). Other external oxidants such as

ferric salts or indigosulfonates have been shown to increase cell division

(2,3). The ubiquitous presence of a transplasma membrane electron transport

system which can reduce external oxidants while transporting protons across

the plasma membrane can be the basis for growth stimulation by impermeable

oxidizing agents (4,5). We can now show that a series of impermeable redox

agents stimulate the growth of HeLa cells in the absence of fetal bovine

serum or other growth factors. Furthermore, other factors which stimulate

serum-free growth increase the rate of electron transport, and antitumor

drugs which inhibit growth inhibit the transplasma membrane redox.

Methods

Hela cells are grown in an atmos here 8 5% CO2, 95% air on a modified minimal essential media (Gibco) at 37 C with 10% fetal hovine serum, 100 u. pencillin and 100 rig/ml streplomycin at pH7.4. Monolayer cultures are

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prepared for study by short trypsin treatment to release cells followed by centrifugation at 15,000 xg. For redox assays the cells are taken up in TD- Tris buffer (NaCl gg/l, KC1 0.3g g/l, Na2 HP04 0.1 g/l and Trizma base 3 g/l, pH 7.4) to a final concentration of 0.2 g cell wet weight/ml. Cells were harvested in expoonential growth phase. For growth assay released cells are counted in a Coulter counter. The same media without serum is used to study oxidant effects. Oxidants are added to the media before innoculation in concentrations ranging from 0.001 to 1.0 mM. Cells are counted after 2 day incubation at 37'. Cell survival is determined by eosin Y exclusion.

Ferricyanide reduction by whole cells is done on an Aminco DW2a spectrophotometer on dual beam at 37' with stirring. TD-Tris buffer is used to suspend 50-200 mg cells in 0.1-0.4 mM ferricyanide. is subtracted from 420 nm.

Absprpl{on ;&500 nm Extinction coefficient is 1 mM cm .

reduction of other oxidants the procedure of Avron and Shavit (6) is used to follow the formation of iron II bathophenanthroline with iron III ammonium sulfate reduction by the added redox agent corrected for control rate with no added redox agent. Iron II bathophenanthroline sulfonate absorbance is measured on the dual beam with subtr cti n of absorbance at 600 nm from 535 nm. Extinction coefficient 17.1 mM -f -P cm . lo-50 mg cells are suspended in TD-Tris buffer with 10 uM bathophenanthroline sulfonate and 3.5 uM ferric ammonium sulfonate plus oxidant at indicated concentration. All reagents must be protected from light to minimize control rates.

Results

Addition of low concentrations of ferricyanide to HeLa cell cultures in

absence of fetal bovine serum or other growth factors stimulates growth to

reach up to one third of the cell count as is achieved with 10% serum. The

maximum effect with ferricyanide is at 0.033 to 0.1 mM. Above this

concentration growth tends to decrease (fig. 1). Ferrocyanide at similar

concentrations has no effect (fig. 1). At the concentrations which stimulate

maximum growth ferricyanide reduction by the HeLa cells is less than maximum

but the rate of ferricyanide induced proton release is saturated at 2 protons

released per ferricyanide reduced (7). Other impermeable redox agents with a

mid-point potential at pH 7.0 of -125 mV or over are reduced by the

transplasma membrane redox system and stimulate growth of HeLa cells to an

extent similar to ferricyanide. No reduction or growth stimulation is seen

with oxidants having a redox potential less than -185 mV. Table I. Internal

oxidants such as pyruvate and other oxycarboxylic acids stimulate melanoma

cell growth (1,8) but pyruvate has very little effect on HeLa cell growth.

(Table I). Insulin also stimulates the growth of HeLa cells in absence of

serum. Ferricyanide gives additional stimulation in the presence of insulin

and the stimulation is seen even at higher concentrations of ferricyanide than

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0 FERMY~~~I~E 0 FEh’ROCYANlOE

0 I 0 0001 00033 0.01 0.033 0.1 0.33

FERRICYANIDE CONCENTRATION (mM)

Fig. 1. Effect of sodium ferricyanide concentration on growth of HeLa cells in absence of serum or other growth factors. Initial innoculum 1.5 x 10’ cells. Ferrocyanide at the same concentration gives no stimulation.

can be used for ferricyanide alone. (Table II). Transferrin also stimulates

HeLa cell growth, but ferricyanide does not give a further increase in the

presence of transferrin (data not shown). We have previously shown that

preincubation of HeLa cells for 3 min with low concentrations of the antitumor

drugs, adriamycin, cisdiammindichloro platinum (cis platin) and bleomycin

inhibit ferricyanide reduction (9,10,11).

Discussion

Transplasma membrane NADH ferricyanide reductase has been demonstrated

in studies with oriented erythrocyte membranes (12,13) and all isolated

plasma membranes examined have NADH ferricyanide reductase activity

(4,14,15). The electron donor for transplasma membrane ferricyanide

reduction by whole cells can therefore be NADH. Ellem and Kay (1) have

pointed out that the growth stimulation of melanoma cells by both

ferricyanide and pyruvate can be based on oxidation of NADH. Since pyruvate

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Table I

Correlation of growth stimulation and rate of reduction of impermeable oxidants with HeLa cells

Oxidants Cont. mM

Growth Stimulation*

Ferricyanide

1,2 Naphthoquinone- 4 Sulfonate

Fe III EDTA

Hexamine Ruthenium III

Indigotetrasulfonate

Indigodisulfonate

Anthraqulnone-1,5-Disulfonate

Anthraquinone-2,6-Disulfonate

Anthraquinone-2-Sulfonate

Pyruvate

0.1 360 53 100%

0.033 187

0.01 94

0.1 51

0.1 - 46

0.033 -125

0.1 -174

0.03 -184

0.1 -225

0.1 -185

17

12

16

15

10

2

2

0

110

90

90

98

97

8

10

0

10

Growth stimulation by ferrlcyanide averages 152 f 68%. Effects of other agents are normalized to ferricyanlde in each experiment so the growth is expressed relative to stimulation observed wfth ferricyanide. Pyruvate is a permeable substrate and is shown for comparison.

does not stimulate HeLa cell growth it appears that only transmembrane

electron transport is sufficient to stimulate growth in these cells.

Furthermore the effective concentration of ferricyanide for growth

stimulation corresponds to the concentrations which gives maximum proton

release (7). Higher concentrations of ferricyanide would increase the rate of

NADH oxidation without increasing proton release. The proton release which

Table II

Effect of ferricyanide on growth of HeLa cells in presence and absence of insulin

Ferricyanide added mM none 0.01 0.033 0.1 0.33

Cell Count per 25 cm2flask x 10e5 No Insulin Plus Insulin

30ug/ml. 0.24 2.4 0.28 2.6 0.52 2.R 1.0 3.1 0.6 4.0

Cells counted after 24 hours growth.

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accompanies transplasma membrane electron transport provides a rational for

growth stimulation since growth has been correlated with increased cytosolic

pH (16,17). We have also shown that transplasma membrane electron transport

in heart can increase internal calcium (18) and changes in internal calcium

have been related to growth stimulation (19,ZO). Since iron III transferrin

can be reduced at the cell surface to release iron II (21,22,23) we postulate

that the transplasma membrane redox system is a transferrin iron reductase.

This would be consistant with the stimulation of growth by transferrin which

is not increased by ferricyanide. Uptake of iron cannot be the only basis for

growth stimulation by ferricyanide. We have previously shown that

ferricyanide is not taken up by liver cells (24). Growth stimulation by

impermeable indigosulfonates or hexamine ruthenium also shows that iron uptake

is not the sole basis for stimulation. Hela cells reoxidize ferrocyanide at

less than 0.01 times the rate of ferricyanide reduction so ferricyanide acts

primarily as an electron sink. With an average cell concentration of 1 x IO5

(0.001 g) per flask ferricyanide would be reduced at 0.1 nmoles min-'. With

400 nmoles per 4 ml flask it would take 4000 min (or 2.7 days) to reduce the

ferricyanide. Thus there is sufficient ferricyanide for the 2 day growth

period. The other acceptors used are autoxidizable in air. Transferrin

catalyses rapid oxidation of iron II as it is reincorporated into the

transferrin (25) so the cycle of reduction and autooxidation of iron in

transferrin is essentially autocatalylic unless the iron is taken up by the

cell. Transferrin may have two roles in growth stimulation. It can act as an

electron acceptor for the transmembrane dehydrogenase (26) and it can supply a

source of iron for uptake from the acid environment of endocytic vesicles.

Three different antitumor drugs (adriamycin, cis platin and bleomycin)

inhibit transplasma membrane ferricyanide reduction at concentrations which

inhibit HeLa cell growth (9,10,11). We propose that growth inhibition by by

these drugs is in response to the inhibition of the transmembrane electron

transport and proton release (10). We have previously shown that the

transmembrane redox system in virus transformed liver cells is more sensitive

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to adriamycin than in untransformed cells (27). Thus growth factors which

stimulate redox stimulate growth, and growth inhihitors can act by inhibition

of the redox system. Modification of the redox system in transformed cells

may allow increased growth with lack of control.

Acknowledgements

Encouragement and support of Professor D.J. Morr& and the Purdue Cancer Center and the advice of Dr. L. Jacobsen in the Cell Culture Center have been essential for the success of this work. Supported in part by grants from the Indiana Elks and the National Science Foundation and a Career Award GMK6-21839 from the NIH to FLC.

References

1.

2. 3.

4.

5. 6. 7.

8. 9.

10. 11. 12.

13.

14.

15.

16.

17.

18. 19.

20.

21. 22. 23. 24.

25.

26.

27.

Ellem, K.A.O. and G.F. Kay (1983) Biochem. Biophys. Res. Communs. 112, 183-190. Brooks, M.M. (1947) Science 106, 320. Rudland, P.S., H. Durbin, D. Clingan and L.J. de Asua (1977) Blochem. Biophys. Res. Communs. 75, 556-562. Crane, F.L., H. LSw, and M.G. Clark (1984) in The Enzymes of Biological Membranes ed. A. Martonosi Plenum, N.Y. 2 edit. Vol. 4. pp. 465-510. Goldenberg, H. (1982) Biochim. Biophys. Acta. 694, 203-223. Avron, M. and l-l. Shavit (1963) Anal. Biochem. 6, 549-554. Sun, I.L., F.L. Crane, C. Grebing and H. Liiw (1984) J. Bioenerg. Biomemb. 16, 315-328. Kay, G.F. and K.A.O. Ellem (1984) Cell and Tissue Kinetics in press. Sun, I.L. and F.L. Crane (1982) Federation proceed. 41, 737. Sun, I.L. and F.L. Crane (1984) Biochem. pharmacol. in press. Sun, I.L. and F.L. Crane (1984) Biochem. Internat. 9, 299-306. Crane, F.L., H.E. Crane, I.L. Sun, W.C. MacKellar, C. Grebing and H. Liiw (1982) J. Bioenerg Biomemb. 14, 425-433. Grebing, C., F.L. Crane, H. LOW and K. Hall (1984) J. Bioenerg. Riomemh. in press. Crane, F.L., H. Goldenberg, D.J. Morre and H. Lijw (1979) in Subcellular Biochemistry ed. D.B. Roodyn, Plenum, N.Y. Vol. 6 pp. 345-399. Goldenberg, H., F.L. Crane and D.J. Morr6 (1979) J. Biol. Chem. 254, 2491-2498. Nuccitelli, R. and D.W. Deamer eds. (1982) Intracellular pH: Its Measurement, Regulation and Utilization in Cellular Function, A.R. Liss, N.Y. pp. 594. Moolenaar, W.H., R.Y. Tsien, P.T. van der Saag and S.W. DeLaat (1983) Nature 304, 645-648. Clark, M.G. (1983) Proceed. Australian Sot. for Medical Res. 16, 5. Mix, L.L., R.J. Dinerstein and M.L. Vfllereal (1984) Biochem. Biophys. Res. Communs. 119, 69-75. Moolenaar, W.H., L.G.J. Tertoolen and S.W. deLaat (1984) J. Biol. Chem. 259, 8066-8075. Morgan, E.H. (1983) Biochim. Biophys. Acta 733, 39-50. Thorstensen, K. and I. Romslo (1984) Blochim. Biophys. Acta 804, 200-208. Cole, E.S. and J. Glass (1983) Biochim. Biophys. Acta. 762, 102-110. Clark, M.G., E.J. Partick, G.S. Patten, F.L. Crane, H. Ltiw and C. Grebing (1981) Biochem. J. 200, 565-572. Bates, G.W., E.F. Workman and M.R. Schlabach (1973) Biochem. Biophys. Res. Communs 50, 84-90. Sun, I.L., F.L. Crane, D.J. Morre and H. Lziw (1985) Federation proceed. in press. Sun, I.L., F.L. Crane, J.Y. Chou, H. Low and C. Grebing (1983) Biochem. Biophys. Res. Communs. 116, 210-216.

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Transplasma membrane redox stimulates HeLa cell growth