The effects of phytohemagglutinin on the growth and histochemical properties of mammalian cells in tissue culture.код для вставкиСкачать
The Effects of Phytohemagglutinin on the Growth and Histochemical Properties of Mammalian Cells in Tissue Culture ' j 2 LOUIS V. CASO Department of Histology, Temple University School of Dentistry, Philadelphia, Pennsylvania ABSTRACT Phytohemagglutinin-P (PHA-P) exhibited inhibitory action on the growth of two human epithelioid cell lines, HeLa and L-132, and on the G929 strain of mouse fibroblasts. Growth of stationary cultures was measured spectrophotometrically by DNA production after 96 hours incubation. The mitotic indices of the three cell lines were reduced significantly by PHA-P after 72 hours incubation. Histochemical studies demonstrate depressed production of cytoplasmic RNA in 72-hour HeLa cultures after treatment with PHA-P, with the appearance of prominent, PAS-positive, diastase-fast granules in the cytoplasm. Bound lipid aggregates increased i n amount in the cytoplasm of HeLa cells after PHA-P treatment. The PAS-positive, diastase-fast granules were also found in the cytoplasm of the L-132 and L-929 cell lines after treatment, but cytoplasmic RNA remained unchanged qualitatively. Alkaline phosphatase was slight in amount in HeLa cells and was reduced to trace amounts after PHA-P treatment. L-132 cells showed moderate to heavy concentration of alkaline phosphatase, which remained unchanged qualitatively after PHA-P treatment. The enzyme could not be demonstrated in the L-929 fibroblasts. Phytohemagglutinin (PHA) is an extract (Rigas and Osgood, ' 5 5 ) of the red kidney bean (Phaseolus vulgaris) which agglutinates erythrocytes in low concentrations and leukocytes in high concentrations. Nowell ('60) observed that PHA causes mitotic activity in the leukocytes of peripheral blood in tissue culture. Mitoses in leukocyte cultures are known to be preceded by the enlargement of small lymphocytes and their transformation to blastoid cells (Carstairs, '62; MacKinney, Stohlman and Brecher, '62). While the mitotic index of normal leukocyte cultures is high after PHA treatment, not all the lymphocytes of an affected culture undergo blastoid cell formation and mitosis (Cooper, Barkhan and Hale, '63), and in certain conditions, such as chronic lymphocytic leukemia (Bernard, Geraldes and Boiron, '64), relatively few or no blastoid cells develop. The stimulatory effects of PHA on leukocytes have been reviewed by Robbins ('64). There is, in addition, evidence for in vivo stimulation of lymphatic tissue by PHA, although other findings indicate suppression of the primary and secondary immune responses in rodents, and inhibition of phagocytosis in the reticuloendothelial sysANAT.REC.,162: 459-466, tem of mice (Lozzio, '67; Jennings and Oates, '67). PHA has been reported to cause increased mitoses in the basal cell layer of human skin explants (Sarkamy and Caron, '65), and acanthosis in guinea pig skin after intradennal injection (Sarkamy and Caron, '66). The findings of Ioachim ('66) indicate that PHA causes clumping of L-929 fibroblasts and other mouse, rat and human cell lines and changes in the morphology of these cell lines in tissue culture. However, the treated cultures were found to be viable and their mitotic indices exceeded those of the controls. In the present study PHA was added to cultures of three established cell-lines : HeLa, derived from a human epidermoid carcinoma of the cervix; L-132, derived from human embryonic lung; and the L-929 strain of mouse fibroblasts. In contrast to the growth-stimulating action seen on lymphocytes, PHA inhibited the growth of these cell cultures and depressed their mitotic indices. 1 Supported by General Research 5-Sol-FR-05339-05. 2This paper was presented in part Session of the American Association at New Orleans, Louisiana, April 10, Support grant at the Annual of Anatomists 1968. 459 460 LOUIS V. CASO MATERIALS AND METHODS D N A cell cultures. All cultures were grown in Blake flasks as stationary monolayer cultures. HeLa and L-929 were cultured in Eagle's minimum essential medium with 15% horse serum and 0.03% glutamine. L-132 cells were grown in LY medium (Difco) with the same concentrations of horse serum and glutamine. Each experiment consisted of a set of PHAtreated cultures and control cultures. The former contained an added amount of phytohemagglutinin-P (PHA-P) (Wilson, '66) in a concentration of either 0.02 ml/ ml, 0.01 ml/ml or 0.005 ml/ml. The controls consisted either of untreated cultures, cultures containing an equivalent amount of triple-distilled water, or cultures containing an equivalent amount of PHA-P suspension resulting from denaturation by heating at 100°C for 15 minutes, or of the clear supernatant from the denatured suspension. Each flask was inoculated with 10' cells/lO ml culture medium, incubated at 36°C for four days, after which the medium was removed from each culture flask and centrifuged. Cells adhering to the glass of HeLa and L-132 culture flasks were lightly trypsinized (0.1 ml 10% trypsin in 5 mlO.85% NaCl), followed by one or two washings of 3 to 5 ml normal saline. L-929 cultures were treated similarly but instead of trypsinization the cells were scraped free from the glass with a rubber policeman. Cells which had been removed from the original medium by centrifuging were added to those recovered by trypsinization or scraping. PHA tended to agglutinate the cells (especially HeLa), which then floated free from the glass. Growth of cell cultures was estimated as a function of total DNA production (Salzman, '59) by the Paul ('56) modification of the micro-method of Ceriotti, using hot perchloric acid extraction and indol color formation. Optical density was read in the Perkin-Elmer spectrophotometer at 489 mu. The O.D. value at 520 mu was subtracted from that at the wave length specific for DNA in order to eliminate interference from substances with wave lengths outside the DNA optical density curve. Histochemical procedures. All cultures were inoculated with 10' cells/lO ml me- dium and grown for 72 hours at 36°C. The culture media were the same as those used in the DNA growth experiments, and all treated cultures contained PHA-P in concentrations stated for specific experiments. (a) RNA. Trypsinized cells were washed three times in Hanks balanced salt solution (BSS) and fixed five minutes in either neutral buffered formalin or Carnoy's fixative, dehydrated and embedded in paraffin (30 to 40 minutes). Paraffi sections were brought to water and incubated in ribonuclease (Pearse, '61), 100 mg/100 ml demineralized water, at 36°C for two hours, accompanied by control sections in demineralized water. Sections were then stained with 1 % toluidine blue. (b) Alkaline phosphatase. The method described by Gomori ('52) and modified by Merchant, Kahn and Murphy ('60) was adapted for use in cell suspensions. The trypsinized cells, washed three times in BSS, were fixed for five minutes in neutral buffered formalin. This was followed by two centrifuge washings in demineralized water. Cell suspensions were reacted in a solution of the substrate (sodium alpha-naphthyl acid phosphate) and pchloro-o-toluidine diazonium salt, and in control solution of diazonium salt alone, for 30 minutes at room temperature, followed by two more centrifuge washings in demineralized water. Cell suspensionswere mounted on microscope slides in glycerogel. (c) PAS reaction. The trypsinized cells were washed three times in BSS and once in normal saline, fixed five minutes in Carnoy's fixative, dehydrated and embedded in paraffin (30 to 40 minutes). Sections were oxidized with 0.5% periodic acid and stained with Schiffs reagent in the usual PAS procedure. To determine if glycogen was present, deparaffinized sections were coated with celloidin and incubated in 1% diastase in neutral buffered saline for one hour at 37°C with suitable control sections. ( d ) Bound lipid. In preparation for the method of Berenbaum ('58) trypsinized cells were washed three times in BSS,fixed for five minutes in Carnoy's fixative, de3 Phytohemagglutinin-P (Difco). approx. 100 mg reconstituted with 5 ml tripledistilled water. 4 Ribonuclease (Nutritional Biochemical Corp. ), crystalline, 5 x. PHA O N TISSUE CULTURE CELL-LINES 461 hydrated and embedded in paraffin. De- tures at 36°C in Leighton tubes till forparaffiized sections were then washed mation of a monolayer on the glass, then overnight in running tap water, dehydrated drawing off the medium and adding, in in absolute acetone and stained in Sudan 2 ml quantities, dilutions of PHA-P, in regBlack B in acetone at 37°C for periods of ular culture medium in concentrations six hours and 24 hours. For the modifica- ranging from 0.0025 ml/ml to 0.02 ml/ tion of the method of Ackerman ('52) ml, followed by observation during the next fresh cell suspensions were used. Whole, 72 hours of incubation. Slight morphologitrypsinized cells were washed three times cal changes, such as withdrawal of some in BSS and fixed five minutes in neutral cytoplasmic processes, or rounding of some buffered formalin. Hydrolysis in 25% cells, were seen at the 0.0025 ml concenacetic acid for five minutes followed, suc- tration, with increased rounding of cells ceeded by two centrifuge washings in de- through the 0.02 ml concentration. Cell mineralized water. Cell suspensions were disintegration increased at the higher conthen stained in Sudan Black B in 70% centrations, but cytotoxicity did not apethanol, followed by four centrifuge wash- pear to reach the plus two grades described ings in 70% ethanol. Cells were mounted by Syverton or by Toplin ('59). HeLa apon microscope slides in glycerogel. peared to be the most resistant to cyto( e ) Baker's pyridine extraction. PHA- toxicity and L-132 the most susceptible. P-treated cultures and control cultures Cell viability was determined by withwere trypsinized, washed three times in drawing the PHA-medium after 72 hours, BSS, centrifuged and fked 20 hours in and adding to the tubes a 1:5000 solution diluted Bouin's fixative according to the of neutral red in sterile Earle's solution. procedure outlined by Pearse ('61 ). Treat- Most cells adhering to the glass were capaed and control cells were extracted 24 ble of showing supravital staining with the hours in pyridine at 60°C. Both treated dye. The range of viability was within the and control cells were washed in tap water, limits, normal for control cultures, 85dehydrated and embedded in paraffin (30- 95% viable cells, for all dilutions of PHA40 minutes) and stained by the PAS pro- P tested. cedure. After the 72-hour treatment of the cells ( f ) Hyaluponidase reaction. Paraffin sec- with the various dilutions of PHA-P, the tions of PHA-P-treated cultures were pre- ability of the cultures to recover normal pared as described for the PAS reaction morphology and growth was tested by rebut were deparaffinized and incubated with moving the PHA-medium, washing the cultesticular hyaluronidase (approximately ture three times with BSS and followed by 100 USP U./ml) in 0.1 M phosphate buf- addition of fresh medium. All cultures fer (pH 7) for 24 hours at 37"C, according regrew normal cells during the first 24 to the method of Gersh and Catchpole hours. ('49) for depolymerization of glycoprotein Mitotic index. The mitotic index of ground substance. Washed sections were each cell-line was determined by counting then stained by the PAS procedure. 1000 cells, in sections embedded in paraf( g ) Other staining reactions. These in- fin and stained with iron hematoxylin, to cluded the Baker modification of the Mil- determine the number of mitotic figures. Ion reaction (Barka and Anderson, ' 6 3 ) , Normal control cultures of L132, L-929 the Nile blue stain of Lillie ('56), iron and HeLa were found to have mitotic inhematoxylin and acid Alcian blue (1% dices of 2.67, 1.7 and 0.96 respectively, in 0.1 N HC1, pH 2). Trypsinized cells and these were reduced in the drug-treated were washed three times in BSS, fixed five cultures, over the range of PHA concentraminutes in Carnoy's fixative, dehydrated tions used above, to 0.87, 0.30 and 0.16 and embedded in paraffin (30-40 minutes). respectively, or one-third to one-sixth the control values. RESULTS D N A growth measurements. It can be Cytotoxicity and cell viability. Prelim- seen in table 1 that PHA-P, 0.02 ml/ml, inary tests for cytotoxicity and viability hyaluronidase (Worthington Biochemwere carried out by incubating the cell cul- ical5 Testicular Corp.). ~~ 462 LOUIS V. CASO TABLE 1 Optical densities a t 489 mp f o r D N A content of HeLa cell cultures (loa cells/lO m l ) a f t e r addition of phytohemagglutinin-P. Incubation at 36" C f o r 96 hours Experiment 1 2 3 4 5 6 7 8 9 10 Control culture (O.D.) PHA-P culture (0.02 mljml) (O.D.) 0.139 0.2001 0.1101 0.232 1 0.2741 0.2281 0.472 0.281 0.2022.4 0.2262.5 0.085 0.098 0.064 0.1272 0.121 2 0.106 0.167 0.176 0.1123 0.138 P < 0.05 < 0.05 < 0.001 < 0.025 > 0.05 < 0.005 < 0.005 < 0.05 < 0.005 < 0.001 lMean value of three cultures. 2 Mean value of two cultures. SMean value of four cultures. 4 Equivalent amount of triple-distilled water added to control culture. 5 Equivalent amount of supernatant from PHA-P (denatured at 100°C for 15 minutes) added to control culture. significantly reduced the growth of HeLa cells. This inhibition was the same whether controls were untreated, or treated by the addition of triple-distilled water or the supernatant from the heat-denatured drug. For lower concentrations of PHA-P (0.01 ml/ml and 0.005 ml/ml) inhibition of HeLa cell growth was suggested but not significant according to the probability value (P) of the Student t test. The L-132 cells proved to be more sensitive to PHA-P, and table 2 shows consistent inhibition at the 0.005 ml/ml and 0.01 ml/ml concentrations. Here it is seen also that the heat- denatured suspension of PHA-P could be used effectively as a control in two experiments. The L-929 fibroblasts exhibited inhibition of growth at the 0.02 ml/ml, 0.01 ml/ml and 0.005 ml/ml concentrations of PHA-P, as shown in table 3. This was consistently significant, except in experiments 9 and 10, in which the controls showed much variability in DNA synthesis. The whole suspension of the heat-denatured drug was used in the controls in these experiments, and perhaps the suspended particles in the culture medium interfered with metabolism at the cell surfaces. When triple-distilled water or the clear supernatant from the heat-denatured PHA was employed in control cultures, the experimental cultures showed consistently significant inhibition of cell growth. Histochemistry. Staining for RNA indicated that 72-hour HeLa cultures, treated with PHA-P, 0.02 ml/ml, showed an even distribution of RNA in the cytoplasm, but intensity of staining was much heavier in untreated control cultures. Qualitatively, four separate experiments indicated a decreased amount of RNA synthesis after PHA-P treatment. Nuclei of control HeLa cells were also much more heavily stained than those of the treated cells, while nucleoli stained the same in both. L-929 and L-132 cell cultures showed heavy cytoplasmic staining for RNA, and this was true for the 0.01 ml/ml and 0.02 ml/ml concentrations of PHA-P. Control cultures of L-929 and G132 cells showed no qualitative difference in RNA staining. TABLE 2 Optical densities at 489 mp f o r D N A content o f human embryonic lung (L-132) cultures (108 cells/lO m l ) a f t e r addition of phytohemagglutinin-P. Incubation at 36'C for 96 hours Experiment Control culture (O.D.) 0.342 0.3461*3 0.395 1A 0.183lr4 0.253 1,4 0.174 0.332 0.2202.4 234 PHA-P culture PHA-P culture ml/ml) ml jml ) (O.D.) (0.005 (O.D.) (0.01 0.1894 0.2463 0.2224 0.1024 0.118 0.100 4 0.160 0.083 P < 0.001 < 0.01 < 0.01 < 0.001 < 0.005 < 0.001 < 0.05 < 0.001 1 Equivalent amount of triple-distilled water added to control culture. 9Equivalent amount of PHA-P (denatured at 100°C for 15 minutes) added to control culture. 3 Mean value for three cultures. 4 Mean value for four cultures. 463 PHA ON TISSUE CULTURE CELL-LINES TABLE 3 Optical densities at 489 mjt for D N A content of mouse fibroblast (L-929)cultures (lo* cells/ 1 0 m l ) after addition o f phytohemagglutinin-P. Incubation at 36°C f o r 96 hours Experiment Control culture (O.D.) PHA-P culture (0.02 ml/ml) (O.D.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0.186 0.372 0.13433' 0.324'J 0.178 s ~ l 0.187L1 0.1984*2 0.3223*2 0.1723 ~ 1 0.2424,' 0.2204.2 0.3525,2 0.2773.2 0.319 52 PHA-P culture (0.01 ml/ml) (O.D.) PHA-P culture (0.005 ml/d) ( O.D. 1 0.1034 0.129 5 0.089 0.162 ' 0.098 0.072 ' 0.114 0.159 0.092' 0.180 0.117 0.2085 0.1883 0.1485 P < 0.025 < 0.025 < 0.025 < 0.001 < 0.005 < 0.05 < 0.01 < 0.01 > 0.2 > 0.2 < 0.001 < 0.05 < 0.05 < 0.001 'Equivalent amount of PHA-P (denatured at 100°C for 15 minutes) added to control culture. Equivalent amount of supernatant from PHA-P (denatured at 100°C for 15 minutes) added to control culture. 3 Mean value of three cultures. 4 Mean value of two cultures. 5 Mean value of four cultures. * Alkaline phosphatase content of HeLa cells qualitatively was diminished in cultures treated with PHA-P, 0.02 ml/ml. In four separate experiments, alkaline phosphatase was distributed in the cytoplasm of most control cells in slight to moderate amounts, and in occasional groups of cells in heavily stained areas, especially just inside the plasma membrane. PHA-P-treated cells showed a similar distribution, but alkaline phosphatase was much more lightly stained even in those areas where it had concentrated more abundantly, and many cells showed no staining or only trace amounts. This was in contrast to the alkaline phosphatase content of C132 cells, which consistently showed heavy cytoplasmic staining both in control cultures, and in those treated with PHA-P at the 0.02, 0.01 and 0.005 ml/ml concentrations. L-929 cells did not stain for alkaline phosphatase, either in control or treated cells. The PAS reaction for polysaccharides demonstrated red cytoplasmic granules in HeLa cells both in control cultures and cultures treated with PHA-P, 0.02 ml/ml. The granules in the treated cells appeared to be coarser and more prominent. Hydrolysis in 1% diastase before PAS staining removed all the red granules from the cy- toplasm of control cells, while many granules persisted in the cells of cultures containing PHA-P. This observation was confirmed in four separate experiments. Both L-132 and L-929 cells showed PASpositive granules in the cytoplasm of cells treated with PHA-P at the 0.02, 0.01 and 0.005 ml/ml concentrations. These granules were finer and present in fewer cells at the lower concentrations of PHA-P, while at the 0.02 ml/ml concentration they were larger and resembled those of the treated HeLa cells. As in the HeLa cultures, the PAS-positive granules of the treated L-132 and L-929 cells also were found to be diastase-fast. Reaction of PHA-P-treated cell cultures with hyaluronidase failed to remove the PAS-positive granules from the cytoplasm. This experiment was repeated with three cultures of L-929 cells at the 0.02, 0.01 and 0.005 ml/ml concentrations of PHAP. The granules were clearly identifiable in all cases, although the culture treated with the 0.02 ml/ml concentration gave somewhat fainter PAS staining than the hyaluronidase control after enzymatic action. L-132 cells, treated with the 0.02 ml/ ml concentration of PHA-P, showed no difference in PAS staining after the hyaluronidase reaction. 464 LOUIS V. CASO Pyridine extraction did not alter the PAS staining of cytoplasmic granules of L-929 cells treated with PHA-P, 0.02 ml/ ml. This experiment was repeated with the L-132 cells also treated with the 0.02 ml/ ml drug concentration, with identical results. Consistent with the negative findings for hyaluronidase and pyridine extraction, the Millon reaction repeatedly failed to demonstrate these granules in the HeLa cells, thus excluding protein, and Alcian blue failed to demonstrate acid mucopolysaccharide granules in either treated or control HeLa cells. Nile blue A failed to demonstrate lipofuscin granules in the same cells. The hydration method of Berenbaum ('58) showed that the cytoplasm of HeLa cells from control cultures and cultures treated with PHA-P, 0.02 ml/rnl, contained fine, rather evenly distributed black granules, indicating bound lipid in both types of cultures in approximately equal amounts. However, the method of Ackerman ('52), modified for use in cell suspensions, demonstrated aggregates of large, black granules in the cytoplasm of many of the cells treated with the 0.02 ml/ml concentration of PHA-P. Some of the control cells had similar granules, but these were comparatively few in number. This would indicate that PHA may induce increased amounts of bound lipid in HeLa cells. Although bound lipid in the oxidized state would be PAS-positive, the granules did not correspond exactly to the PAS-positive granules described in the PHA-Ptreated cells. DISCUSSION The cytochemical changes in leukocytes after PHA stimulation have been investigated rather extensively, and it is known that alkaline phosphatase decreases in amount in polymorphonucleocytes after PHA treatment, and degeneration of these cells proceeds more quickly. The "blast" cells of PHA-stimulated cultures are substantially negative for alkaline phosphatase (Quaglino, Hayhoe and Flemens, '62). After PHA treatment, RNA synthesis has been found to begin within the first few hours, and DNA synthesis in mononuclear cells in 24-48 hours (Killander and Rig- ler, '65). Uridine-5-3H labeling suggests that RNA synthesis occurs first in the nucleus and later in the cytoplasm in stimulated leukocyte cultures (Winter and Yoffey, '65). There may be a difference in the type of RNA molecule in lymphocytes stimulated by PHA and bacterial antigen, the former stimulating non-ribosomal RNA and the latter stimulating a precursor to ribosomal RNA (Cooper and Rubin, '65). The transformation of lymphocytes and the accompanying RNA and DNA changes by PHA are similar to those described more recently for another stimulator of lymphocyte metabolism, pokeweed mitogen, which however produces two types of stimulated lymphocytes, one of which possesses PASpositive, diastase-resistant granules. ( Chessin et al., '66; Schwarz, '68). In addition, it should be noted that PHA, in high concentrations, depresses lymphocyte transformation and their uptake of labeled thymidine (Wilson, '66). Glycogen has been found to accumulate in transforming lymphocytes prior to DNA synthesis (Hayhoe and Quaglino, '65). It reaches a maximum in lymphocytes at 1824 hours and persists for 48 hours. Most "blast" cells are negative, their energy for transformation having been derived from glycogen which was then depleted (Quaglino, Hayhoe and Flemens, '62). Razavi ('66) found that a fluoresceinlabeled PHA moiety localized in the cytoplasm of lymphocyte "blast" cells and granulocytes. It is possible that the mammalian cells showing PAS-positive granules in the present study behave like the granulocytes of PHA-stimulated cultures. The possibility that these granules represent incompletely metabolized PHA-P in the cytoplasm merits further investigation. The results of the present study, using diastase, pyridine extraction and the hyaluronidase and Millon reactions, would seem to exclude glycogen, glycolipid, and glycoprotein from constituting the substance of the diastase-fast granules, although some glycogen granules were present in the treated HeLa cells. The agglutinating and mitogenic actions of PHA can be differentiated by heating (Holland and Holland, '65) or erythrocyte absorption (Barkhan and Ballas, '63), and possible separate factors for agglutination and mitogenic action have been indicated PHA O N TISSUE CULTURE CELL-LINES by electrophoresis (Marshall and Norins. ' 6 5 ) . PHA, absorbed by erythrocytes, exhibits agglutination of S-180 cells, an action distinct from erythrocyte agglutination (Tunis, '64). Furthermore, unabsorbed PHA-P has been shown to agglutinate many cell types, including erythrocytes and leukocytes of various species, C3H sarcoma, Ehrlich ascites tumor and L fibroblasts (Steck and Wallach, '65). These cells have one or more binding sites in common with PHA. Changed electrophoretic mobilities of lymphocytes treated with PHA suggest adsorption of the drug (Vassar and Culling, '64). In view of these findings, the inhibitory action of PHA-P on HeLa, L-929 and L-132 cells may be due to interference of the agglutinating factor of PHA at the plasma membrane. Stimulation of lymphocyte '%last" cell formation by PHA is similar to stimulation in vitro of previously sensitized lymphocytes by antigen (Benezra, Gery and Davies, '67) or to stimulation of lymphocytes in the homograft reaction (Gowans, McGregor, Cowen and Ford, '62), but the difference in the RNA molecule produced by antigen and PHA indicates a different mechanism of action for each (Cooper and Rubin, '65). However, the mitogenic action of PHA is neutralized by antiserum to PHA-M, the substance itself being antigenic (Byrd, Finley, Finley and McClure, '64), and human splenic extract inhibits or suppresses the transformation of leukocytes by PHA (Pegrum, '65). The stimulating effects of anti-leukocyte antiserum on leukocytes resemble the action of PHA in potency and time sequence (Grasbeck, Nordman and de la Chapelle, '64). In the present study, PHA possibly may be acting like an antibody (agglutinin) by coating antigenic sites on the plasma membrane and thereby interfering with cellular metabolism. The resemblance of PHA stimulation of lymphocytes to antigenic stimulation, and the ability of PHA to agglutinate cells other than blood cells by a separate agglutinating factor, suggest a dual action which should be investigated further. If the agglutinating factor is indeed responsible for the inhibition of cell growth in tissue culture, the possibility of its selective inhibitory action on certain cells, while lymphatic 465 cells are either stimulated or relatively unaffected, suggests potential therapeutic value. ACKNOWLEDGMENT The author expresses his appreciation and thanks to Mrs. Nevart Gulezian and Miss Helen Ruane for their technical assistance. LITERATURE CITED Ackerman, G. A. 1952 A modscation of the Sudan black B technique for the possible cytochemical demonstration of masked lipids. Science, 115: 629-631. Barka, T.,and P. J. Anderson 1963 Histochemistry, Theory, Practice and Bibliography. Harper and Row, New York, 45-46. Barkhan, P., and A. Ballas 1963 Phytohemagglutinin: separation of hemagglutinating and mitogenic principles. Nature, 200: 141-142. Benezra, D.,I. Gery and A. M. Davies 1967 Transformation of rabbit lymphocytes by specific antigens. Proc. SOC.Exp. Biol. Med., 125: 1305-1308. Berenbaum, M. 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