a-difluoromethylornithine alters calcium signaling in ... · [cancer research 52, 6782-6789,...

[CANCER RESEARCH 52, 6782-6789, December 15, 1992]

a-Difluoromethylornithine Alters Calcium Signaling in Platelet-derived Growth

Factor-stimulated A172 Brain Tumor Cells in Culture I

Burt G. Feuerstein, 2 J~nos Szollosi , Hirak S. Basu, and Laurence J. Marton

Brain Tumor Research Center of the Department of Neurological Surgery [B. G. F., H. S. B.], Division of Molecular Cytometry of the Department of Laboratory Medicine lB. G. F., L. J. M.], and Department of Pediatrics [B. G. F.], School of Medicine, University of California, San Francisco, California 94143, and Department of Biophysics, Medical University School of Debrecen, Nagyerdei krt 98, 1t-4012 Debrecen, Hungary [J. S.]

ABSTRACT

a-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h hut less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism.

INTRODUCTION

All mammalian cells contain the polyamines putrescine, spermidine, and spermine and tightly regulated pathways for their synthesis (1). Polyamine levels and the activities of their rate-limiting biosynthetic enzymes increase up to 100-fold when quiescent cells are stimulated to proliferate (2). Interfer- ence with polyamine accumulation results in inhibition of cell growth (1). Because of the importance of these compounds in cellular growth, drugs that interfere with their biosynthesis have attracted interest as potential antineoplastic agents. DFMO, 3 a specific irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, is being tested as a prophylactic against colon and skin cancer (3-7) and as a treatment for several tumors, including primary brain tumors (8).

Glioblastoma multiforme, the most common primary brain tumor in humans, is believed to derive from oligodendrocyte type 2 astrocytic cells, which are regulated by PDGF produced by type 1 astrocytes (9, 10). Glioblastoma cell lines often express mRNA of PDGF A and B chains (11-15); the B chain is closely related to the v-sis oncogene (16-18). The identification of PDGF A and B chains and PDGF receptors in human brain

Received 4/16/92; accepted 10/2/92. The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants CA 13525, CA 37606, CA 49409, and CA 41757 from the NIH, by the National Brain Tumor Foundation, and by Grants OTKA 1492 and OKKFT 1142 from the Hungarian Academy of Science.

2 To whom requests for reprints should be addressed, at The Editorial Office, Department of Neurological Surgery, 1360 Ninth Avenue, Suite 210, San Fran- cisco, CA 94122.

3 The abbreviations used are: DFMO, a-difluoromethylornithine; PDGF, platelet-derived growth factor; NMDA, N-methyl-D-aspartate; [Ca2+]i, intracellular calcium concentration; tdeb delay time; teh~, channel opening time; EGTA, ethyleneglycol bis(/~-aminoethyi ether)-N,N,N',N'-tetraacetic acid; HEPES, 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid.

tumor specimens and cell lines (11, 14, 19) suggests the exist- ence of an autocrine loop involving the PDGF signaling pathway.

PDGF stimulates a cascade of cellular events that culminates with entry into the cell cycle and eventual cell division (20). The initial interaction of ligand PDGF with the extracellular domain of its receptor triggers activities in its intracellular do- mains, including enzymatic phosphorylations and release of second messengers (21-23). These events result in release of internal calcium stores, opening of the plasma membrane channels, and elevation of the free [Ca2+]i (24-27). Since PDGF regulates growth and development in the astrocytic lineage, these initial events are all possible regulatory targets of polyamines in astrocytic brain tumors.

The effects of polyamines on the initial stages of PDGF signaling have not been determined. Several investigators have reported interactions between polyamines and intracellular calcium. Spermidine and spermine influence mitochondrial (28-30) and sarcoplasmic reticulum calcium transport (31). Fast receptor-mediated increases in ornithine decarboxylase reportedly raise intracellular polyamine concentrations and alter cellular calcium fluxes. The simultaneous addition of the signaling agent and DFMO reversibly disrupts the increase in polyamines, the changes in calcium fluxes, and cellular re- sponses to the agent (32, 33).

In these experiments, we studied the effect of D F M O on calcium signals produced by PDGF in A172 human glioblastoma cells. Our goal was to determine whether polyamine depletion caused by DFMO alters the sources of calcium used by the cell for its initial calcium signal and whether such changes are reversed by polyamine repletion.

MATERIALS AND M E T H O D S

Reagents and Chemicals. PDGF BB homodimer and DFMO were provided by Dr. Glenn Pierce (Amgen, Inc., Thousand Oaks, CA) and Marion Merrell Dow (Cincinnati, OH), respectively. Putrescine, spermidine, and spermine were from Calbiochem (La Jolla, CA). Indo- I-AM was purchased from Molecular Probes (Eugene, OR). MnCI2 and EGTA were from Sigma (St. Louis, MO). All other chemicals were of reagent grade.

Cell Culture. Mycoplasma-free A 172 human glioblastoma cells were frozen at the onset of these experiments; every 3 months, a new aliquot was used. Cells were seeded at --~20,000/cm 2 and grown to confluence in Dulbecco's Modified Eagle's Medium-H21 supplemented with glutamine and 10% fetal calf serum at 37~ in a humidified atmosphere containing 5% CO2. Cells grown for spectrofluorometry were seeded on glass coverslips and placed in plastic Petri dishes.

DFMO Treatment and Polyamine Repletion. The cell cultures were depleted of polyamines by treatment with 5 mM DFMO for 1-7 days. DFMO was added after a full day of growth to avoid treating lag phase cells. Polyamines were repleted by adding 1 mM putrescine to DFMO- treated cells. For faster repletion kinetics, 1 mM putrescine and 50 UM spermidine were added in the presence of 1 mM aminoguanidine.

Colony-forming Efficiency. The colony-forming efficiency was determined as previously described (34). Briefly, A 172 cells were added to

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CALCIUM SIGNALING AFTER PUTRESCINE AND SPERMIDINE DEPLETION

50,000 heavily irradiated, preincubated autologous feeder cells and grown for 15 days. After fixation and staining, colonies consisting of at least 50 cells were counted. The plating efficiency was calculated as the ratio of colonies formed to cells seeded.

Polyamine Analysis. Cells were washed twice with phosphate-buff- ered saline, placed in 8% sulfosalicylic acid, sonicated, and kept at 4~ for 1 h. The acid-soluble material was derivatized with dansyl chloride, loaded onto a CIs reverse-phase column, and separated by elution with a gradient of acetonitrile and water at 500C (35). The polyamines, analogues, and metabolites were detected and quantitated with a Per- kin-Elmer LS4 fluorescence spectrometer and an LCI-100 computing integrator.

Preparation of Cells for PDGF Stimulation. Cells were prepared for calcium measurement as previously described (36, 37). Briefly, confluent A172 cells were serum starved for 3-6 h, loaded with indo-l-AM ester for approximately 1 h at 37~ and washed with and placed in 37~ HEPES buffer (20 mm HEPES, 123 mm NaCI, 5 mm KCI, 1.5 mm MgCl2, 1 mM CaCl2, 5 mm sodium pyruvate, pH 7.2). After equilibra- tion at 370C, the cells were stimulated with various concentrations of PDGF. In some experiments designed to investigate the calcium source for the PDGF-stimulated calcium signal, EGTA or Mn 2+ was also added. For cells monitored in suspension, 2- to 3-ml aliquots were incubated with the dye for 60-90 min, washed twice, and resuspended in HEPES buffer at a density of 0.5-1.0 x 106 cells/ml.

Laser Fluorescence Image Cytometry. The fluorescence intensity of intraceUular indo-1 was mapped on an ACAS 470 laser image cytometer (Meridian Instruments, Okemos, MI) as previously described (36, 37). Slide chambers (Nunc, Inc., Naperville, IL) were mounted in a humidified, temperature-controlled chamber attached to the computer-controlled stage of an inverted microscope. During scanning, the cells were moved by the computer-controlled stage. Subcellular loca- tions were excited through a quartz objective by a 350-360 nm beam from an argon ion laser. The results reported here were obtained in the line scan mode. Emitted fluorescence was directed by a 380 nm short pass dichroic mirror to a 450 nm dichroic mirror where the light was split and passed through 405 + 20 nm and 485 + 20 nm band pass filters for simultaneous ratio analysis by two photomultipliers (all filters were from Omega Optical, Brattleboro, VT). At times, a 420 + 20 nm band pass filter was used to monitor the isoemissive point of the equilibrium between free indo-I and calcium-bound indo-l. A calibration curve for calcium concentration was derived by determining the ratio of indo-1 fluorescence in solutions of known calcium concentration opti- mized with glycerol to simulate the cytoplasmic environment (38, 39).

Ca 2+ Channel Opening and Closing. Fluorescence at the isoemissive point of the equilibrium between the indo-I calcium complex and free indo-1 (which is directly proportional to the total amount of dye in the scanned field) was monitored at 420 nm, and the emission maximum of free indo dye was monitored at 485 nm with the filters noted above; 400-600 mm MnCI2 was added to the medium at appropriate times. Because Mn 2+ quenches the fluorescence of indo-1, open calcium channels were distinguished by a drop in the fluorescence intensity at the isoemissive point in the presence of the ion (37).

To measure the time between intracellular calcium release and calcium channel opening (tcha,), MnCI2 was added before PDGF. The technique of measuring calcium channel opening is illustrated in Fig. I. When intracellular calcium is released after PDGF is added, the fluorescence of free indo dye falls, but the fluorescence at the isoemissive point remains stable. This indicates that free dye has been bound by the increasing numbers of calcium ions. When the fluorescence at 420 nm decreases, Mn 2+ has entered the cell and has quenched the dye. In this example, the calcium channels opened approximately 40 s after intracellular calcium was released (about 80 s after addition of PDGF). To determine whether calcium channels were open after stimulation by PDGF, MnCI2 was added at various times after PDGF.

Spectrofluorometry. The results in single cells were confirmed by spectrofluorometry and flow cytometry in populations of A172 cells. The spectrofluorometric measurements were obtained with an SLM 8000 fluorescence spectrophotometer as previously described (36). In- do-l-loaded cells on coverslips were held in HEPES buffer in temper-

4oooo T

~ 30000 i

oooo

~ 10000 M n P[](~F ~'~"~,~.L .~

0 50 100 150 200 250 300

Time (sec) Fig. l. Measurement of calcium channel gating after PDGF stimulation.

MnCI2 (300 #M) and PDGF (20 ng/ml) were added at the arrows. The figure shows indo-1 fluorescence at 485 nm (upper trace, beginning at ~35,000) and 420 nm (lower trace, beginning at ~ 22,000). After addition of PDGF, fluorescence at 485 nm decreased as intracellular calcium stores were released (small arrowhead), while fluorescence at 420 nm remained constant. Calcium channels opened when the intensity at 420 nm fell (large arrowhead).

ature-controlled cuvette. Dye in the cells was excited by 360 nm light through an 8 nm slit, and emitted light was collected simultaneously through a monochromator at 405 nm (8 nm slit) and through a 485 _+ 20 nm band pass filter by two photomultipliers. The ratios of these fluorescence intensities were computed and stored at 2-s intervals by an IBM PC. Calibration was performed on these same cells as previously described (36, 40).

Flow Cytometry. Suspensions of indo-l-loaded cells were analyzed in a Becton Dickinson 440 flow cytometer at 37~ by using a 350-360 nm beam from an argon ion laser. Forward angle and orthogonal light scatter, the fluorescence intensities at 405 + 10 nm and 485 + 10 nm, the ratio of these intensities, and time were monitored. The mean ratio was determined as a function of time; the percentage of responding cells was also determined as a function of time by finding the percentage of cells with ratios greater than 2 SD above the mean base=line cell ratio. This system was calibrated as previously described (36, 41).

R E S U L T S

Effects of D F M O on Cell Growth and Polyamine Levels. Assays of colony-forming efficiency showed that D F M O was not toxic to A 172 cells (data not shown). Growth curves showed that D F M O had little effect when cells were seeded at numbers close to confluency (Fig. 2). Putrescine was depleted after 1 day of D F M O treatment, and spermidine was depleted after 2 days of treatment; spermine was not depleted even after 7 days of t reatment with D F M O (Fig. 3).

Effect of D F M O on PDGF-induced Calcium Signals in Sin- gle A172 Cells. [Ca2+]i over time in a representative control A172 cell s t imulated with 20 ng/ml P D G F is shown in Fig. 4A. Base-line [Ca2+]i was near 130 nm, and P D G F was added at ---75 s. [Ca2§ increased above base line at --+100 s. The tde~ between addit ion of P D G F and init iat ion of the calcium signal was ---25 s. The peak [Ca2+]i was near 420 nM. After peaking, the calcium signal decreased quickly and then plateaued, but did not return to base line by the end of the experiment. In recent experiments in which cells were monitored longer, PDGF-s t imulated calcium signals in control cells lasted 700-1600 s (data not shown).

[Ca2+]i over time in a cell that responded to P D G F with a full calcium signal after 4 days of t reatment with D F M O is shown in Fig. 4B. Base-line [Ca2+]i was near 110 nM, P D G F was added

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C A L C I U M S I G N A L I N G A F T E R P U T R E S C I N E A N D S P E R M I D 1 N E D E P L E T I O N lO8] D F M O P U T

1 0 s

1 0 4

0

i l i

2 4 6

T i m e [ d a y s ]

Fig. 2. Growth of A172 cells. Cells were treated with DFMO and later with putrescine (PUT). DFMO inhibited growth only slightly. Control ([2); DFMO (5 raM) (A); DFMO (5 mM) and putrescine (1 mM) (A). If not shown, errorbars are less than symbol size.

shown in Fig. 5. Control cells and cells treated with D F M O for 4 days exhibited similar decreases in tdel as a function of the P D G F dose. The peak height, percentage of responding cells,

_~ and duration of response varied directly with the P D G F dose. The peak height did not differ significantly between control and DFMO-trea ted cells, but the calcium signal duration was longer in control cells. Base-line [Ca2+]i was not significantly affected by D F M O (data not shown). Cells replete with putrescine and spermidine more often responded to P D G F with a complete calcium signal than did cells depleted of these compounds. Treatment with D F M O for 15 min to 3 days did not shorten the calcium signal (data not shown).

Effects of D F M O on PDGF-induced Calcium Signals in Pop- ulations of A172 Cells. The PDGF-induced calcium signals from confluent populat ions of control and DFMO-trea ted cells attached to glass coverslips are shown in Fig. 6. Approximately 25 s after s t imulation with 20 ng/ml PDGF, the ratio of emission intensities increased to peaks near 0.065 in both samples.

!

0 In control cells, the ratio gradually decreased but did not reach the original base line by 700 s. DFMO-trea ted cells reached a plateau value slightly higher than the base line at approximately 300 s. The addition of EGTA decreased the ratio more in control cells than in DFMO-trea ted cells.

Flow cytometric results are shown in Fig. 7. In control cells, as P D G F dose increased from 0.5 to 100 ng/ml, the tale] decreased from about 75 to 25 s, and the peak height of responding cells increased from about 150 to 500 nM. Fewer than m

"~== 2.5 ] DFMO + ~f PUT

~=-= o.s A

k O "1

0.5

0.3

3 o . 2

~ 1 ~ ~ " : " I : : " I " I " I " " : [ : " " I " " " I " : " I : : " 1 ~" �9 2 0 0 4 0 0 6 0 0 8 0 0

=~ E 0 ' & "' Time ( s e c )

w

o ~ _~=

L. O

2.0

1.0

0.0 . . . . . . - ~- 0 2 a 6 8

Time (Days)

Fig. 3. Polyamine levels in A172 cells. Putrescine (PUT) and spermidine levels decreased in DFMO-treated cells. Addition of putrescine increased spermidine and putrescine levels to or above control levels. Control ([]); DFMO (A); DFMO + PUT (A).

at --~ 125 s, the tde~ was near 35 s, and the peak [Ca2+]i was near 300 riM. The total durat ion of the calcium signal was approximately 300 s.

P D G F dose-response relationships for tdel, peak [Ca2+]i, durat ion of response, and percentage of fully responding cells are

0 . 5 - -

v 0 . 4 -

0 . 3

0.2-

0.1-

0 0 200 400 600 800

Time (see) Fig. 4. A, [Ca2+]i in a control A172 cell. The signal did not return to base line

by the end of the experiment. B, [Ca2+]i in a DFMO-treated A172 cell. The calcium signal was considerably shorter in the control cell than in the DFMO- treated cell.

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1


. . . . . . . . ii

1 0

6O0

5O0

r v

__.= 400 o

-r.

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30O

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. . . . . . . ! . . . . . . . !

1 0 1 0 0

1 O0 800

7OO 80

6OO

60 i ~ 4o

0 4 0 0

2 0 3 0 0

0 2 0 0

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POGF dose (ng/ml) PDGF (ng/ml)

Fig. 5. PDGF dose response for delay time, peak height, percentage of responding cells, and duration of response in control (ll) and DFMO-treated ([]) cells. A full calcium response was defined as a signal lasting longer than 75 s. Bars, 1 SD.

30% of cells in any control sample continued to respond 300 s after signaling began. In DFMO-treated cells, as the PDGF dose increased from 2 to 100 ng/ml, the tde I decreased from 50 to 25 s, peak height increased from about 300 to 450 riM, and fewer than 30% of cells continued to respond 200 s after signaling began.

Release of Intracellular Calcium and Calcium Channel Gat- ing in DFMO-treated Cells. Because PDGF-induced calcium signals include both release of intracellular calcium stores and opening of plasma membrane calcium channels (37), alterations in either of these events might cause shortened calcium signals. Experiments utilizing extracellular EGTA to remove extracellular calcium provided information on intracellular calcium release. There were no significant differences between control and DFMO-treated cells in the length (99 -+ 21 v e r s u s 114 + 26 s, respectively) or peak height (270 _+ 73 v e r s u s 280 + 54 nm) of the calcium signal produced by intracellular calcium release measured in the presence of EGTA.

The time between release of intracellular calcium and opening of plasma membrane calcium channels (tchan) w a s

40 _+ l0 s in control and 42 _+ 16 s in DFMO-treated cells. Therefore, DFMO did not affect the timing of calcium channel opening.

The gating of calcium channels in fully responding control and DFMO-treated cells is shown in Table 1. In control cells, the calcium channels were open if [Ca2+]i w a s above base line and closed if it had returned to base line, whereas in DFMO- treated cells, the calcium channels remained open even if [Ca2+]i had returned to base line. The channels closed at nearly the same time in control and treated cells (after 600 s).

Polyamine Repletion and Calcium Signal Length. Within 1 day after the addition of 1 mM putrescine to A172 cultures, intracellular putrescine was higher than control, and spermidine was restored nearly to control levels, but spermine was not affected (Fig. 3). The duration of the PDGF-induced calcium signal returned to the control value within 1 day (data not shown). When putrescine and spermidine were quickly restored to levels above control by adding them to cells after DFMO treatment, the calcium signal reached control length 10-24 h after polyamine addition.

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O

O

a:

0.10

0.08

0.04

0"02 t ~ PDGF EGTA ~ ~Ca 0.00 , - , .

0 200 400 600 800

Time [sec]

0.10"

0.08

0.06

0.04

0.02

~ ~ q ~ - ~'---- ~ -..---- ----~ - -~- -:: --~.-~T

PDGF ~ EGTA 0.08

0 200 400 600 800

Time [sec] Fig. 6. [Ca2+]i determined by spectrofluorometry in A 172 cells after stimula-

tion with 20 ng/ml PDGF. ,4, control cells. B, cells treated with 5 mM DFMO for 4 days. PDGF, EGTA, and calcium were added where noted.

DISCUSSION

This study has shown that 4 days of treatment with 5 mM DFMO shortens the PDGF-induced calcium signal in A172 human glioblastoma cells. This effect was reversed by adding putrescine for more than 10 h. DFMO did not alter the colony- forming efficiency, base-line [Ca2+]i, intracellular calcium release, or the opening and closing of plasma membrane calcium channels. These findings suggest that DFMO exerts a specific effect on the calcium signal induced by PDGF.

A shortened calcium signal must ultimately result from a decrease in the amount of intracellular free Ca 2+. In a previous study, we found that calcium signals in A172 cells derive from intracellular Ca 2+ stores and from Ca 2+ entry through plasma membrane channels, and that intracellular release precedes the opening of plasma membrane calcium channels (37). To determine whether disordered intracellular calcium release contrib- utes to the shortened calcium signals we observed, we chelated extracellular calcium with EGTA before stimulation with PDGF. Under these conditions, DFMO-treated and control cells showed no differences in the peak height and length of the calcium signal. We therefore conclude that DFMO did not affect intracellular calcium release.

Monitoring with a manganese-quenching technique (37) showed that DFMO did not qualitatively affect either the opening or the closing of plasma membrane calcium channels. Thus,

DFMO-induced shortening of the calcium signal did not appear to be caused by differences in their gating.

These findings suggest that calcium extrusion mechanisms are targets for DFMO in A172 cells. One possibility for regulation is a Na*/Ca 2+ exchanger and a second is a calcium pump. Neither has yet been studied in this cell line. In vitro, polyamines have been found to inhibit the ATPase activity of a plasma membrane calcium pump (42). Polyamines also affect the activities of protein kinase C (43-45) and calmodulin (46) and interact with cytoskeletal proteins and membrane phospho- lipids (47); all of these substances regulate the activity of membrane-associated calcium pumps (48-53) or calcium exchangers (54, 55), and may form a basis for the effects we observed in this study.

A regulatory site for polyamines on the NMDA receptor, a Na t , K § Ca 2§ gated channel, has been described (56, 57). Both spermidine and spermine enhance NMDA-induced Ca 2§ cur- rents in cells that express this receptor, and DFMO inhibits expression of NMDA-induced injury (58). This channel is blocked by Mg 2+ until membrane depolarization, when the receptor complex can be activated by NMDA or glutamate (59). However, neither activator was added during our experiments and therefore NMDA receptors are not likely to be responsible for our results.

Intracellular calcium stores are another possible source of polyamine-induced alterations in [Ca2+]i . Spermine and spermidine reportedly stimulate mitochondrial calcium uptake (28- 30) and inhibit isolated sarcoplasmic reticulum calcium release (31). If mitochondrial uptake is inhibited or if endoplasmic reticular stores are more easily released, [Ca2+]i should increase. We found no evidence for DFMO-induced increases in [Ca2+]i in these experiments.

Koenig et al. (32, 33) hypothesized that polyamines are second messengers involved in early events just after cellular stimulation, and found that inhibiting polyamine biosynthesis with DFMO reversibly inhibited calcium uptake. We found no alterations in PDGF-induced calcium signaling immediately after DFMO treatment. These contrasting results may be caused by differences either in methods of calcium measurement or in the agonists and tissues used.

The relationships between polyamine concentrations and calcium signaling in our polyamine repletion and depletion experiments were equivocal. Depletion of putrescine and spermidine took less than 1 day and 1-2 days, respectively, whereas shortening of the calcium signal only appeared after 3 days of DFMO treatment. The DFMO-induced shortening of the calcium signal was reversed within 1 day after addition of putrescine, at which time the putrescine concentration was higher than the control level and spermidine and spermine concentrations were near control levels. Attempts to quickly replete putrescine and spermidine by adding them simultaneously did not lead to a corresponding quick reversal of shortened calcium signals. It took more than 10 h but less than 24 h to regain normal calcium signaling; within 2 h, however, the putrescine and spermidine concentrations were 4 to 5 times higher than control. Thus, the intracellular polyamine concentrations did not correlate with calcium signaling in either the depletion or the fast repletion experiments. It is possible that polyamine concentrations vary in different cellular compart- ments, and therefore gross intracellular levels may not reflect the concentrations at target sites. It is also possible that the abnormally high intracellular polyamine concentrations in the

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o

100

60

40 0

[Ca2+]i

I ~-- 700 nM 100 ] i DGF C A PDGF - ~ 500 nM ~ 80

~ 2o

I ~--- 100 nM , i i i 0 100 200 300 400 500 0 100 200 300 400 500 600

Time [sec] Time [sec]

[Ca2+]i

I 100 j PDGF D B ~-- 700 nM 100 PDGF ,--,

500 nM ~ 80

so 60

~- - 100 nM I , , i i

0 100 200 300 400 500 0 t . . . . . Time [sec] 0 loo 200 300 400 soo 600

Time [sec] Fig. 7. [Ca2+]i (.4, B) and percentage of responding cells (C, D) in control and DFMO-treated A172 cells stimulated with various doses of PDGF. In C and D, a

response was defined by an increase greater than 2 SD above resting [Ca2+]i. A, 5 ng/ml PDGF (control), 2 ng/ml PDGF (DFMO-treated); A, 5 ng/ml PDGF; O, 20 ng/ml PDGF; O, 1 O0 ng/ml PDGF.

fast repletion experiments might have prevented a return to control levels of calcium signaling.

Although the PDGF-induced calcium signals were shortened in single, attached DFMO-treated A172 cells and in populations of attached and unattached cells, there were quantitative differences in the results. These differences arise from the tech- niques themselves (36). In single attached cells, the peak height, delay time, and length of the calcium signal are easily determined because fluorescence intensities emitted from specific cells of interest can be monitored through the microscope. In cell populations, however, these measurements are more diffi- cult to make. In spectrofluorometric studies, the fluorescence intensity emitted from every dye molecule excited by the inci- dent beam is measured. In the best case, the dye is intracellular and homogenously distributed throughout the cell population. However, if dye leaks out of cells, the intracellular measurements are skewed by an extracellular calcium concentration 2-3 orders of magnitude higher than the [Ca2+]i . Thus, the rapid decrease in fluorescence intensity ratio after EGTA treatment in DFMO-treated cells measured by spectrofluorometry could indicate either changes in [Ca2+]i brought about by chelation of extracellular Ca 2+ or prior leakage of indo dye into the extracellular fluid followed by Ca 2§ chelation. Because our image cytometric studies of single cells did not reveal altered [Ca2+]i in response to EGTA at this point in the calcium signal

6787

and because we saw evidence of indo-1 leak in DFMO-treated A172 cells (data not shown), we conclude that there was a significant leak of indo-1 acid into extracellular fluid in this sample.

The apparent [Ca2+]i might also be skewed toward cells that take up more dye, because they will contribute more emission

Table 1 PDGF-induced calcium channel closing in .4172 cells MnCI2 was added to cell cultures at various times after stimulation with

PDGF. Only data from cells that responded fully to PDGF are included. Calcium channels were considered open if fluorescence intensity at 420 nm decreased rapidly upon addition of Mn 2+.

Cells with channels Time after PDGF Cells with [Ca2+]i open (n)/cells stimulation when above base line (n)/ measured (n) MnCl2 was added (s) cells measured (n)

Control 7/7 80 7/7 2/2 140 2/2 7/7 200 7/7 0/6 770 0/6

DFMO 8/8 60 8/8 8/8 380 0/8 6/6 440 0/6 6/6 550 0/6 7/7 570 0/7 1/5 630 0/5 7/8 745 0/6 4/13 860 0/13




in tens i ty than cells t ak ing up less dye. H o w e v e r , in our s tudies

in single cells, we found no ev idence of this p h e n o m e n o n (data no t shown) . In add i t ion , cell p o p u l a t i o n s may inc lude sub-

g roups o f cells tha t do no t r e spond to the s t imulus , dec reas ing

the value o f peak [Ca2+]i . Th i s e r ro r was no t large e n o u g h to m a k e a s igni f icant d i f ference in our m e a s u r e m e n t s .

T h e ca l c ium signals assayed by f low c y t o m e t r y were shor te r

t han those m e a s u r e d by image c y t o m e t r y or spec t ro f luo rome- try. T h e P D G F - s t i m u l a t e d ca l c ium signals in con t ro l cells were

400 s by f low c y t o m e t r y and over 700 s by image cy tome t ry , whe reas in D F M O - t r e a t e d cells, the s ignals were 200 and --~ 300 s, respect ively. T ryps in by i tself does no t sho r t en the ca lc ium signal; however , t ryps in ized cells in con tac t wi th a surface have

longer ca l c ium signals t han t ryps in ized cells in suspens ion (36). Bo th con t ro l and D F M O - t r e a t e d cells in suspens ion had

shor t e r ca l c ium signals t han those w h i c h were a t tached . The re - fore, the m e c h a n i s m s by w h i c h D F M O shor t ens ca l c ium t ran- s ients m u s t differ f rom those in u n a t t a c h e d cells.

In conc lus ion , P D G F - i n d u c e d ca l c ium s ignal ing is disor- de red in the A172 h u m a n g l iob la s toma cell l ine af ter dep le t ion o f pu t resc ine and s p e r m i n e by D F M O . H o w e v e r , in t race l lu la r ca l c ium re lease and p l a s m a m e m b r a n e ca l c ium c h a n n e l open-

ing and c los ing do no t a p p e a r to be affected. This leaves ca l c ium ex t rus ion as a possible m e c h a n i s m . These a l t e ra t ions in signaling m a y con t r ibu te to the g rowth - inh ib i to ry effects o f D F M O . To evaluate this possibil i ty, we are s tudy ing the r e l a t ionsh ip o f ca l c ium s ignal ing to D N A synthes is in s ingle cells.

A C K N O W L E D G M E N T S

The authors thank Phillip Hsieh, Amgen, Inc., for purifying the PDGF; Griffith R. Harsh IV for providing Mycoplasma-free A172 cells; Harrihar Pershadsingh, G. Vereb, and M. Das for helpful discussion; Warren Lubich for polyamine analyses; and Stephen Ordway for editorial assistance.

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1992;52:6782-6789. Cancer Res Burt G. Feuerstein, Jànos Szöllösi, Hirak S. Basu, et al. Cells in CulturePlatelet-derived Growth Factor-stimulated A172 Brain Tumor

-Difluoromethylornithine Alters Calcium Signaling inα

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