Inhibition of phytohemagglutinin-, periodate- and A23187-induced lymphocyte transformation by Vinca alkaloids

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Experimental

INHIBITION

Cell Research 124 (1979) 413-420

OF PHYTOHEMAGGLUTININ-,

AND A23 187-INDUCED

LYMPHOCYTE

BY VINCA JOHN C. SCHULTZ,

PERIODATETRANSFORMATION

ALKALOIDS

JEAN M. MARTIN

and NASROLLAH

T. SHAHIDI

Department of Pediatrics, University of Wisconsin, Madison, WI 53792, USA

SUMMARY The effect of the Vinca alkaloids, vincristine and vinblastine, on mitogen-induced transformation of isolated human peripheral blood lymphocytes has been investigated. Cells were subjected to a variety of mitogens (PHA, ionophore A23 187and sodium periodate) whose mechanism and site of action diier. Addition of vincristine or vinblastine to lymphocyte cultures prior to mitogen produced a concentration-dependent inhibition of cell transformation as determined by measurement of DNA synthesis and blast formation. The inhibitory effects were not due to decreased cell viability, since the drugs had little or no effect on cell viability. Vincristine and vinblastine were also found to imnair rSHlthvmidine incorooration by mestimulated blast cells at the higher drug concentrations t&ted: The results presenied in this communication show that the Vincaalkaloids block lymphocyte transformation induced by either lectin or non-lectin mitogens. This suggests that the inhibitory step(s) may occur after mitogen stimulation.

Transformation of T-lymphocytes can be [12, 131 have shown that the microtubuleinduced by a variety of non-specific mito- disrupting agents, vincristine (VCR), vingenie agents, including phytohemagglutinin blastine (VLB) and colchicine suppress the (PHA) [l, 21, concanavalin A (ConA) [3- appearance of ConA-induced blasts in 5], periodate [6-8], sequential treatment mouse splenic lymphocytes, rabbit and huwith neuraminidase followed by galactose man peripheral blood lymphocytes and muoxidase [9], and the calcium ionophore rine lymphocytes from mouse spleen. Medrano et al. [15] have subsequently shown A23187 [lo, 111. It is generally thought that specific inter- that colchicine, vinblastine and cytochalaactions between certain cell surface recep- sin B (CB) inhibit human peripheral blood tors and the lectin mitogens (PHA and lymphocyte transformation induced by ConA) are responsible for the initiation of PHA. In contrast, stimulation of neuraminievents leading to cell proliferation and dase-treated lymphocytes by galactosetransformation. It is also thought that direct oxidase is not inhibited by colchicine at or indirect interactions between these spe- concentrations that inhibit the mitogenic response induced by ConA [ 161.Stenzel et al. cific cell surface receptors (glycoproteins) and microtubules may be connected with [17] reported that a 30 min exposure of huthe initial events of lectin-induced blasto- man peripheral blood lymphocytes to colchicine potentiates the mitogenic effect of genesis [12-141. sodium periodate on the cells suggesting a In this regard, Edelman and co-workers 27-791815

Exp Cell Res 124 (1979)

414

Schultz., Martin and Shahidi

selective stimulatory effect of colchicine on Mononuclear cell isolation Peripheral venous blood was obtained from healthy lymphocyte responses induced by cell-cell donors and collected in preservative-free heparin (20 contact. U/ml blood). Lymphocytes were immediately isolated by centrifugation over Ficoll-Hypaque at 400 g In view of the above, it has been postu- for 30 min [ 181. The lymphocyte-rich suspension was lated that the Vinca alkaloids and related carefully harvested from the interface, washed by rein PBS; recentrifuged and counted in a drugs inhibit mitogenesis by disruption of suspension hemocytometer. The mononuclear cell preparations the microtubule system of the lymphocyte. contained an average of 92 % lymphocytes and 8% as judged by Wright staining and an averSince the lectin mitogens (PHA and ConA) monocytes age of 12 platelets per nucleated cell. The mononubind to specific cell surface glycoprotein re- clear cell preparations also contained a small number ceptor sites it is not clear whether the in- of red cells. These unseparated mononuclear cells were used throughout this study, unless mentioned hibitory action of Vinca alkaloid is medi- otherwise. ated through modification of cell surfacePeriodate studies binding sites or microtubular structures. Periodate oxidation was nerformed bv incubatine; IvmThe present investigation was designed phocytes at a concentraiion of 20x iOg cells/ml?n-0.4 to ascertain whether non-lectin mitogens, mM NaIO,/O.Ol M sodium ohosnhate (DH 7.4)/0.15 M NaCl for 3’0 min at 4°C. The reaction ‘was terminated which do not require the presence of spe- by centrifugation at 1000 g for 10 min at 4°C and cific binding sites for their action, would resuspension once in PBS, pH 7.2, and then in RPMI1640. The cells were susoended at a final concentraalso be unable to induce blastogenesis in tion of 1x 106cells/ml in.RPMI-1640 containmg fetal the presence of Vinca alkaloids. The use of calf serum (FCS) (10%. heat inactivated at 56°C for 30 and supplemented with penicillin (100 U/ml), non-lectin mitogens would allow us to ex- min), streptomycin (100 &ml), and amphotericin B (0.25 plore directly the role of the microtubules &ml). Cultures of 1 ml were prepared in triplicate polystyrene tubes (12x75 mm) loosely capped, and in the cell transformation process. The in incubated at 37°C in a humid atmosphere of 5 % CO,/ Vinca alkaloid’s interference with lympho- air. Twenty-five ~1 of [3H]thymidme (2.5 &i) were added to ceU cultures (1 ml) that had been incubated cyte transformation may lie on step(s) sub- for 48 h. After additional incubation of the cell culsequent to initial stimulation and be totally tures for 4 h, the cells were collected from the tubes Reeve Angel glass fiber filter paper (934 AH, non-specific as to what agent or technique onto 1x28 inches, Whatman Incorp., Clifton, N.J.) using a semi-automatic microharvesting device. The filters is used to induce transformation.

MATERIALS

AND METHODS

Reagents Vincristine sulfate (VCR). vinblastine sulfate (VLB), and A23 187were generous gifts from Eli Lilly and Co: ; Indianapolis, Ind. PHA (Type V) was obtained from Sigma, St Louis, MO. Sodium metaperiodate was obtained from Fisher Scientific Co., Fair Lawn, N.J. [Methyl-SH]thymidine (55-65 Cilmmole) was purchased from New England Nuclear, Boston, Mass. Cell culture media, antibiotics, and fetal calf serum (FCS) were obtained from Grand Island Biological, Grand Island, N.Y. The serum was heat inactivated at 56°C for 30 min prior to use. A stock solution of A23187 was prepared in absolute ethanol (2.2 mg/ml). Microliter amounts were added to water with rapid mixing. Aliquots of this solution were added to the cell cultures at the desired concentration. Vincristine and vinblastine were prepared in phosphate-buffered saline (PBS). AU solutions were sterilized by Millipore filtration prior to use. Exp Cdl Res 124 (1979)

were washed 6 times with 1.5 ml aliquots of 0.15 M NaCl and air-dried. The cell associated radioactivity was counted in 2 ml of a scintillation fluid consisting of 5 g of 2,5-diphenyloxazole (PPG) and 0.1 g p-bis[2-(5phenyloxazolyl)]-benzene (POPOP) dissolved in 1 1 of toluene and counted in a Packard Tri-Carb scintillation spectrometer (Model 3375). The mean cpm of triplicate cultures was determined and the degree of stimulation was expressed where indicated as the stimulation index (SI) which is the ratio of cpm of stimulated or experimental cultures/control cultures (minus mitogen). The counting efficiency for tritium was 34%.

-

A23187 studies Cells were suspended for culture in RPMI-1640 supplemented with penicillin (100 U/ml), streptomycin (100 &nl), amphotericin B (0.25 &ml) and 10% FCS. Cultures of 1 ml (1 x 1Oecells) were prepared in triplicate in polystyrene tubes (12x75 mm). A23187 (1.1 pg) was added to the tubes in a volume of 20 ~1. The tubes were loosely capped and incubated for 72 h at 37°C in a humid atmosphere of 5% COJair. Cultures were pulsed for 4 h with 2.5 @i of [JH]thymidine. harvested and counted.

Effects of Vinca alkaloids on lymphocyte

Table 1. Elfect of vincristine peripheral

on PHA-induced

stimulation

transformation

of lymphocytes

415

isolated from

blood

The values shown in parentheses correspond to the percentage of r3H]TdR incorporation, taking as 100% that obtained in the presence of mitogen alone

Additions None PHA alone PHA plus 1x 1O-7M VCR 1x lo-@M VCR 1x 1O-5M VCR

VCR added prior to PHA stimulation” Incorp. radioact. (mean cpm _+S.D.)

VCR added after maximal PHA stimulation* Incorp. radioact. (mean cpm _+SD.)

7 9912885 168 69716 000 (100)

7 795+1 846 135 684f14 112 (100)

77 55254 470 (46) 75 568k2 572 (45) 49 331+739 (29)

136 411f3 751 (100) 150 687+21 256 (111) 89 773+6 864 (66)

a Vincristine was added to the cell suspension in a volume of 25 ~1, 4 h prior to addition of 50 ~1 of PHA (0.6 pg). After 72 h of incubation with PHA at 37”C, 25 ~1 of r3H]TdR (2.5 &i) were added and incubation continued for an additional 4 h at 37°C. * Fifty ~1 of PHA (0.6 fig) was added to the cell suspension and the cells cultured for 72 h at 37°C. Vincristine was then added in a volume of 25 ~1 and the cells cultured for an additional 4 h after which 25 ~1 (2.5 &i) of r3H]TdR were added and the incubation continued for an additional 4 h at 37°C.

PHA studies Cells were suspended for culture in RPMI-1640 supplemented with penicillin (100 U/ml), streptomycin (100 &g/ml), amphotericin B (0.25 pg/ml) and 10% FCS. Cultures were done in flat-bottom microtiter plates (Linbro Scientific Co., New Haven, Conn.). Each well contained 0.2 ml of cell suspension (2X 105 cells) and each set of cultures consisted of an unstimulated control culture. PHA (0.6 pg Sigma Type V) was added to the wells in a volume of 50 ~1. All volumes were adjusted to a final volume of 0.25 ml with Medium (minus FCS) and the cultures were incubated for 72 h. Twenty-five ~1 of rH]thymidine (2.5 &i) were added to cell cultures (0.25 ml) and mixed well. After additional incubation of the cell cultures for 4 h, the cells were collected from the wells onto glass fiber filters, washed and counted.

Cell viability Viability of cells after exposure to all concentrations of VCR and VLB was monitored by their ability to exclude trypan blue dye (Grand Island Biological Co., Grand Island, N.Y.).

RESULTS Effect of vincristine on PHA-induced blastogenesis and on uptake of r3H]TdR by transformed peripheral blood lymphocytes

The results of several experiments indicating the effect of various concentrations of vincristine (1 x IO-’ M to 1x 10e5 M) on

PHA-stimulated lymphocyte proliferation measured at 72 h are shown in table 1. Vincristine was added to lymphocyte cultures after maximal stimulation with PHA to measure the effect of vincristine on transformed cells uptake of [3H]TdR and prior to PHA when investigating its effect on the PHA-stimulated transformation of the human peripheral blood lymphocytes. Vincristine was present throughout the culture period. When vincristine was added to cultures at a final concentration of 1x 10e5M, 72 h after PHA stimulation, there was a 34% decrease in [3H]TdR incorporation compared to cultures when vincristine was omitted. 1x 10e7M and 1X 10e6M vincristine were without effect on the uptake of C3H]TdR by the prestimulated cells. Addition of vincristine 4 h prior to PHA resulted in the following changes in proliferative response. (Vincristine and vinblastine were introduced into cultures of lymphocytes 4 h prior to addition of PHA or A23187 when studying the effect of the drug on lymphocyte transformation since it was observed that inhibition of lymphocyte transformaExp Cell Rcs 124 (1979)

416

Schultz, Martin and Shahidi

Table 2. Effect of vincristine phocytes

on periodate-induced

stimulation

of peripheral

blood lym-

Following periodate oxidation of the cells as described under Materials and Methods and suspension of washed oxidized cells in RPMI-1640 (10% FCS), 1 ml aliquots (1 x 106cells) were transferred to culture tubes. The values shown in parentheses correspond to the percentage of rH]TdR incorporation, taking as 100% that obtained in the presence of mitogen alone

Additions None Periodate alone Periodate plus 1x lo-’ M VCR 1x 1O-BM VCR 1x 1O-5M VCR

VCR added prior to culturing of periodate stimulated cells” Incorp. radioact. (mean cpm + S.D.)

VCR added after maximal periodate stimulationb Incorp. radioact. (mean cpm IL SD.)

2 69Ok401 53 542f3 160 (100)

2 116+228 58 324+3 502 (100)

10 9502 1 300 (20) 4 937+114 (9,‘ ’ 3 883f371 (7)

52 125+211 (89) 42 64721 670 (73) 36 11721 861(62)

a Vincristine (in a volume of 100 ~1) was added to one set of the oxidized cells. The drug treated and untreated cells were incubated for 48 h at 37°C. After 48 h, 2.5 &i of [WJTdR in a volume of 25 ~1 was added and incubation continued for another 4 h at 37°C. b Vincristine of the appropriate concentration in a volume of 100 ~1 was added to the oxidized cells that were incubated for 48 h at 37°C in the absence of the drug. The cells were incubated with VCR for 4 h. Twentyfive ~1 of [WjTdR (2.5 /Xi) were added to the cultures and incubation was continued for another 4 h at 37’C.

tion by the drugs measured after 72 h with [3H]TdR incorporation was greater when compared with cultures when drug and mitogen were added simultaneously.) At a vincristine concentration of 10 PM, there was a 71% decrease in [3H]TdR incorporation on comparison with control cultures (minus vincristine). Vincristine concentrations of 0.1 and 1.0 PM depressed the incorporation of [“H]TdR 54 % and 55 %, respectively. Substitution of VLB for VCR in the experiments shown in tables l-3 resulted in very similar responses and therefore are not shown. Effect of vincristine on periodateinduced blastogenesis and on uptake of [3H]TdR by transformed peripheral blood lymphocytes

Normal human lymphocytes respond maximally to NaIO, oxidation when treated at a concentration of 0.4 mM NaIO, (pH 7.4) at 4°C for 30 min. The peak of the NaIO, response occurred at 48 h. Control cells Exp Cell Res 124 (1979)

(minus NaIO,) were also maintained at 4°C for 30 min prior to incubation at 37°C for 48 h. The results of several experiments indicating the effect of various concentrations of vincristine (1 x lob5 M to 1X 10e7M) on periodate-induced lymphocyte proliferation measured at 48 h are shown in table 2. When vincristine at a concentration of 1X 10v5 M was added after 48 h of culturing oxidized cells there was a 38 % suppression of r3H]TdR incorporation. An 11 and 27% decrease in [3H]TdR incorporation were observed at 1x 10v7 M and IX lo+ M VCR, respectively. Addition of VCR immediately after oxidation (prior to 48 h culturing) resulted in the following changes in proliferative response. At a VCR concentration of 1x IO+’ M there was a 93 % inhibition of [3H]TdR incorporation. Decreases of 91 and 80 % in C3H]TdR incorporation were observed at 1x low6 M and 1x lo-’ M, respectively . The periodate-induced stimulation index

Effects of Vinca alkaloids on lymphocyte

Table 3. Effect of vincristine

on A23187-induced

stimulation

transformation

of peripheral

417

blood lym-

phocytes The values shown in parentheses correspond to the percentage of [3H]TdR incorporation with the value obtained in the absence of vincristine taken as 100%

Additions None A23 187alone A23187 plus 2x lo-’ M VCR 2x 1O-6M VCR 2x lo-+ M VCR

VCR added prior to A23 187 stimulation0 Incorp. radioact. (mean cpm + SD.)

VCR added after maximal A23187 stimulatiorP Incorp. radioact. (mean cpm + SD.)

6 344+50 27 857k352 (100)

5 120+1 540 32 706+ 1 887 (100)

13 576f 1 035 (48) 12 391kl 267 (44) 6 673f427 (24)

34 490+ 1 904 (105) 26 108fl 132 (80) 20 071f2 712 (61)

o Vincristine (in a volume of 100 1) was added to the culture tubes containing the lymphocyte suspension 4 h prior to addition of 20 ~1 of A23187 (1.1 pg). After 72 h of incubation with A23187 at 37”C, 25 ~1 of [3H]TdR (2.5 PCi) were added and incubation continued for an additional 4 h at 37°C. Control tubes (minus vincristine) contained 100~1 of PBS. b Twenty ~1 of A23187 (1.1 pg) were added to the culture tubes containing the lymphocyte suspension and the cells cultured for 72 h at 37°C. Vincristine was then added in a volume of 100 /.LI and the cells cultured for an additional 4 h after which 25 ~1 (2.5 &i) of rH]TdR were added and incubation continued for an additional 4 h at 3PC. Control tubes (minus vincristine) contained 2 ~1 of absolute ethanol.

of peripheral blood lymphocytes in the absence of drug was in the range of 1S27. Effect of vincristine on A23187-induced blastogenesis and on uptake of C3H]TdR by transformed peripheral blood lymphocytes

Our results show that the calcium ionophore A23187 induces optimal peripheral blood lymphocyte transformation, asjudged by the increased rate of [3H]TdR into DNA, when the cells are treated with the ionophore at a concentration of 1.0 @g/ml at 37°C for 72 h (not shown). The stimulation index of A23187 response in the absence of vincristine is in the range reported by other workers [lo, 111. The results of several experiments indicating the effect of various concentrations of vincristine (2x lo-’ M to 2~ 10m5M) on A23 187-stimulated peripheral blood lymphocyte proliferation and uptake of [3H]TdR by cells prestimulated with A23 187 are shown in table 3. The effect of increasing concentrations of VCR on the ability of

A23 187-transformed cells to incorporate [3H]TdR into DNA resulted in a progressive inhibiting response. r3H]TdR incorporation was depressed 20 % at 2 x low6 M and 39 % at 2~ 10e5M VCR concentrations. Vincristine at a concentration of 2~ lo-’ M had very little effect. Addition of vincristine to cultures prior to A23 187-stimulation resulted in the following changes in proliferative response. At a vincristine concentration of 2 x lo+ M there was a 76 % inhibition of [“H]TdR incorporation. At 2~10-~ M and 2x10-’ M VCR, 56 and 52% decreases respectively, in thymidine uptake occurred. Blast formation in lymphocytes stimulated with periodate, A23187 or PHA in the presence of vinblastine or vincristine

In order to determine whether these substances are able to effect blastogenesis they were added at various concentrations to cell cultures 4 h before addition of PHA and A23 187 and immediately following washing Exp Cell Res 124 (1979)

418

Schultz, Martin

and Shahidi

Table 4. Blast formation in lymphocytes the presence of vinblastine or vincristine

Additions None Mitogen alone Mitogen plus Vinblastine at 2X lo-’ M 2x lo+ M 2X lO-5 M Vincristine at 2X lo-’ M 2X lo+ M 2X lo+ M

stimulated

with periodate,

A23187 or PHA in

Periodate

A23187

PHA

PHA

Percentage blast transformation

Percentage blast transformation

Percentage blast transformation

Percentage [3H]TdR incorporation (mean cpm + S.D.)

6: (100)

3: (100)”

8: (lOO)U

117 10 0645129 854rt6 971 (lOO)*

12 (18) 6 (9) No discemible cells

19 (56) 8 (23) 1 (3)

43 (49) 22 (25) 20 (23)

37 057k3 585 (31) 24 585+548 (21) 25 608f518 (22)

15 (23) 7 (11) No discemible cells

13 (38) 4 (12) 3 (9)

10 (11) 10 (11) 12 (14)

23 569+ 1 038 (20) 28 145+1 057 (24) 28 123+1 019 (24)

D The values shown in parentheses correspond to the percentage of blast formation taking as 100% that obtained in the presence of mitogen alone. * The values shown in parentheses correspond to the percentage of [3H]TdR incorporation with the value obtained in the absence of the drug taken as 100%. Periodare oxidation. 25x 106 peripheral blood lymphocytes isolated by the Ficoll-Hypaque method were subjected to NaIO, oxidation as described under Materials and Methods. The washed oxidized lymphocytes were suspended in 25 ml RPM&l640 (10% FCS). One ml aliquots (1X1@ cells) were transferred to culture tubes. Vincristine or vinblastine of the desired concentration were added to the cell suspension in a volume of 0.1 ml. These additions were made as soon as the cells were washed and transferred into the culture tubes. Tubes were mixed gently and the cells cultured for 48 h at 3PC. PHA and A23187 stimulafion. Peripheral blood lymphocytes (1 X lo6 cells) in a 0.8 ml volume of RPMI1640 (10% FCS) were incubated with the inhibitors 4 h prior to addition of mitogen. PHA (2.4 pg) contained in a volume of 0.2 ml RPMI-1640 (10% FCS) was added and the cells cultured for 72 h. The PHA-containing tubes were run five times. At the end of 72 h two tubes were used for measurement of DNA synthesis as described in Materials and Methods. A23 187 (1.1 pg) in a volume of 20 ~1 was added to 1.O ml cell cultures (1 X 106cells/ml) RPMI-1640-10% FCS. The mixture was vortexed and the cells cultured for 72 h. (1) A cytocentrifuge smear was made of the cell suspension and stained with Wrights stain. A 100 cell differentiated count was performed by the same observer throughout the study. Blastic transformation was judged on the basis of increased cell size, multiple nucleoli, cytoplasmic basophilia and characteristic nuclear staining. (2) Twenty-five ~1 of rH]TdR (2.5 PCi) was added and the cells cultured for an additional 4 h at 37”C, harvested and the cell associated radioactivity determined.

and suspension of the periodate-oxidized cells. Their effect on blast transformation was observed at the maximal stimulation response times of PHA, A23187 (72 h) and periodate (48 h). To determine if blast formation was inhibited in a parallel fashion to DNA synthesis, the PHA-stimulated cultures were simultaneously monitored for [3H]TdR incorporation into DNA. Vincristine and vinblastine are porent inhibitors of the blastic transformation. At the highest Vinca alkaloid concentration tested (2 x 10m5 Exp Cell Res 124 (1979)

M) the periodate-treated cells were viable, but severely distorted making any differentiation impossible. It is apparent that DNA synthesis and blast transformation are inhibited in a similar fashion. The difference in the number of blast cells in cultures with and without VCR or VLB represents inhibition by the drugs of the transformation reaction of small resting lymphocytes to large proliferating blastoid cells. A small population of cells escape the drug’s action on blastogenesis.

Effects of Vinca alkaloids on lymphocyte

transformation

419

The resulting blast cells’ ability to incorporate [3H]TdR, however, is as shown in tables l-3 adversely affected by the presence of the drug.

A23187 mitogenic activity is also affected. In the inhibition of PHA-stimulated reaction by colchicine and vinblastine, the effect of the drugs does not seem to lie primarily on blocking PHA binding to the lymCell viability phocyte membrane but rather acts on subThe percentage of viable cells in cultures sequent steps [15]. In addition, Greene et incubated with and without Vinca alkaloid al. [19] have shown that 10T5M colchicine (VCR or VLB) measured after 72 h were as and 10T5 M vinblastine did not alter lz51follows: 95-100% in the absence of Vinca ConA binding to human peripheral blood alkaloid; 98-99 % at 10e6M Vinca alkaloid; lymphocytes. Ranney 8z Pincus [20] have 94-100% at 10e5M; and 93-97 % at 10v4M. recently reported suppression of stimulatWe conclude that the inhibitory effects of ing cell activity in mixed lymphocyte culthe VCR and VLB cannot be attributed to ture response reactions by colchicine and vinblastine. decreasing cell viability. The question of the drugs inhibiting DNA synthesis or thymidine transport of the cells DISCUSSION which may account for part of the deWe have been able to confirm the inhibi- pressed transformation reaction was partion of PHA-induced lymphocyte trans- tially answered by incubating the drugs with formation by Vinca alkaloids. In addition, blast cells from maximally stimulated culthe above data demonstrate that sodium pe- tures formed in the absence of the drugs. riodate, a chemical oxidizing agent and cal- Significant decreases in C3H]TdR incorporacium ionophore A23 187-induced lympho- tion by peripheral blood PHA-transformed cyte transformation are blocked by Vinca cells was observed at the highest VLB and VCR concentration tested (1 x lop5 M). The alkaloids. Since lymphocyte transformation by the above mitogens involve different inhibitory effect of the drugs on r3H]TdR mechanisms, it is conceivable that the tar- incorporation by lymphoblasts may result get area of the drugs may lie in blockage of from (a) impairment of thymidine transport subsequent step(s) following the initial of subsequent reactions leading to DNA stimulation by mitogen. Since the complete synthesis; (b) extensive morphological alsequence of events leading to increased cell terations of the cells similar to that observed with human leukemic lymphoblasts proliferation and transformation following mitogen stimulation has not been estab- [21] and erythrocytes [22]. A dissociation of the inhibitory effect of lished, it is difficult to predict the precise mode of inhibitory action of the Vinca al- the drug on lymphocyte transformation kaloids. Whether this event(s) is dependent from the decreased uptake of r3H]TdR by on an intact microtubule-like structure re- prestimulated cells can be observed at the lower vincristine and vinblastine concentramains to be established. In the lectin-stimulated reactions, the tions. At low concentrations, the Vinca Vinca alkaloids may interfere with the bind- alkaloids inhibit the lectin and non-lectining of lectin to the specific surface receptor induced lymphocytes and have little effect sites, but clearly the drugs action is not on [3H]TdR incorporation by transformed limited to this alone since periodate and cells. However, at high concentrations Exp Cell Res 124 (1979)

420

Schultz, Martin and Shahidi

these drugs also inhibit the incorporation of [3H]TdR in already transformed lymphocytes. Several groups have shown that the mitogenie effects of A23 187, PHA and ConA are dependent upon the presence of extracellular Ca2+ [23-261. An increased influx of 45Ca2+has been reported to occur within minutes of PHA binding to human blood lymphocytes [22]. db-cGMP enhances the 45Ca2+uptake induced by ConA while dbCAMP is inhibitory [25]. The mitogenic action may thus involve a critical Ca2+influxdependent reaction(s). It remains to be established whether the Vinca alkaloids interfere with Ca2+metabolism which is so interrelated with the process of lymphocyte transformation and microtubular structure. We thank Mrs Judy Setzkom for her skillful technical assistance with the assay of blast transformation. This work was supported in part by grants from the NIH (CA0536 and GM07131).

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Exp CellRes 124 (1979)

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25. Freedman. M H. Raff. M C & Gomaerts. B. Nature (London) 255 (1975) 378. 1 ’ 26. Parker, C W, Biochem biophys res commun 61 (1974) 1180. Received April 19, 1979 Revised version received June 28, 1979 Accepted July 11, 1979