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Decreased phytohemagglutinin receptor sites in chronic lymphocytic leukemia
SHORT COMMUNICATIONS
542 BBA 23 552 Decreased phytohemagglutinin
receptor
sites in chronic lymphocytic
leukemia When lymphocytes from normal human subjects are incubated with phytohemagglutinin, they transform into large cells capable of undergoing mitosis. In contrast, lymphocytes from patients with chronic lymphocytic leukemia show both a delayed and an impaired blastogenic response to phytohemagglutininl-4. The mechanism for this defective responsiveness has not been elucidated. Since the binding of phytohemagglutinin to cell surface receptor sites appears to be the initial step in the sequence of events leading to mitogenesis, we have examined normal and chronic lymphocytic leukemic lymphocytes in order to determine whether a defect in chronic lymphocytic leukemic lymphocytes could be localized at this step. Highly purified phytohemagglutinin was prepared from Bacto-Phytohemagglutinin-P (Difco Laboratories) by SE-Sephadex chromatography as described by WEBER et aL5. The material in Peak III (ref. 5) was further purified by gel filtration on Sephadex G-150 to give a protein with potent erythroagglutinating as well as lymphocyte stimulating properties. This protein, after being iodinated by the method of AWAI AND BROWNS, retained its full erythroagglutinating activity. Lymphocytes were isolated as follows: For every 85 ml of fresh heparinized blood 15 ml of Gy,6 dextran in saline were added and the red cells were allowed to settle at 37’. The supernatant fluid was freed of granulocytes by passage through a column of nylon fibers packed into a 5o-ml glass syringe. The filtrate was sedimented twice at 400 x g for 7 min, cold distilled water was added to the cell pellet to osmotically lyse the remaining red blood cells, and after 25 set, enough 3.474 saline was added to restore isotonicity, and the suspension was centrifuged for 7 min at 400 x g. The white cell pellet containing go--100% lymphocytes was suspended in Eagle’s minimal essential media7 at a concentration of about 20.10~ cells per cm3. The final cell suspensions contained platelet per 2 lymphocytes.
less than I red cell per IOO lymphocytes and I or fewer The binding of phytohemagglutinin to lymphocytes was
not affected by the osmotic shock step. Total cellular sialic acid was determined by heating intact cells at 100’ for 1.5 min in I M HCl (ref. 8). The cellular debris was removed by centrifugation and the released sialic acid was measured by the method of WARRENS. Enzyme releasable sialic acid was determined in the same way after lymphocytes were incubated in minimal essential medium containing 50 units/ml of neuraminidase (Calbiochem Corp.) for I h at 37’. The binding reactions were carried out in plastic counting tubes in minimal essential medium which contained in 0.4 ml : from 1.25 to 12.5 pg [1311]phytohemagglutinin (specific activity 6.25.10~ counts/min per pg), 2 mg bovine serum albumin, and 2.106 lymphocytes. After 30 min incubation at room temperature, the reaction had come to equilibrium and 6 ml of saline were added, the cells were sedimented, washed with another 6 ml saline, and then the bound radioactivity was determined in a Packard Autogamma counter. Appropriate corrections were made for nonspecific binding to the plastic tubes (in the absence of cells) which accounted for less than 5 y0 of the total counts bound. The data were plotted as described by STECK AND Biochim.
Biophys.
Acta,
192 (1969) 542-545
SHORT COMMUNICATIONS
543
The amount of [1311]phvtollemagglutinin bound was directly proportional ” to the number of lymphocytes present. When the ability of normal and chronic lymphocytic leukemic lymphocytes to
WALLACH~.
bind [1311]pllytohemagglutinin was measured as a function of [1311]phytohemagglutinin concentration, it was found that chronic lymphocytic leukemic lymphocytes bound significantly less phytohemagglutinin than did normal lymphocytes. Typical binding curves are shown in Fig. I. In all three instances the apparent dissociation constant (K) for phytohemagglutinin bindingwas approximately the same (8.4--10.4,~g phytohemagglutinin per ml), while the maximal amount of phytohemagglutinin bound per 2.10~ lymphocytes differed considerably (1.48 pg by the normal lymphocytes compared to 0.36 and 0.80 ,ug by the chronic lymphocytic leukemic lymphocytes. Lymphocytes from 12 normal donors had a mean K (* S.E.) of 12.0 f 1.1 peg phytohemagglutinin per ml while lymphocytes from 14 patients with chronic lymphocytic leukemia had a mean K of 10.3 f 0.7. These values are not significantly different (P > 0.1). In contrast, the lymphocytes from the 12 normal donors bound on the average 1.16 f 0.08 ,ug phytohemagglutinin per ~2.10~ cells while the chronic lymphocytic leukemic lymphocytes (14 donors) bound only 0.49 f 0.06 pg phytohemagglutinin per 2. IO6 cells, an amount significantly less than the normal (P < 0.001). If the molecular weight of the phytohemagglutinin is assumed to be 128000 (ref. IO), one can calculate that the average number of receptor sites per normal lymphocyte is 2.7.10~ and per chronic lymphocytic leukemic lymphocyte, 1.15.10~. This estimate compares with 3.4.10~ receptor sites per erythrocyteii and 6.6.107 receptor sites per Ehrlich ascites carcinoma celllo. The amount
of phytohemagglutinin
bound per chronic
lymphocytic
leukemic
I.2
Fig. I. The binding of phytohemagglutinin (PHA) to lymphocytes. Normal and chronic lymphocytic leukemic lymphocytes were incubated with [1311]phytohemagglutinin as described in the text. The data have been plotted (B) by the method of STECK AND WALLACH~’ according to the equation : C [PHA
bound] = &
’ [PiA]
+ i
where [PHA] = concentration free phytohemagglutinin (,@. n = number of PHA binding sites per cell expressed as pg phytohemagglutinin bound per lymphocyte, and C = number of cells. The dissociation constant K for maximal phytohemagglutinin binding to lymphocytes was determined from the I/[PHA] intercept and the pg phytohemagglutinin bound per lymphocyte, n, from the C/[PHA bound] intercept. o-0, normal donor; o-0, chronic lymphocytic leukemic donor with a peripheral lymphocyte count of r4 roo/mms; A-A, chronic lymphocytic leukemic donor with a lymphocyte count of 54 5oo/mm3. Biochim.
Bioflhys. Acta, I92 (1969) 542-545
544
SHORT COMMUNICATIONS
lymphocyte is inversely related to the patient’s peripheral lymphocyte count (Fig. 2) ; thus, the higher the patient’s lymphocyte count the fewer ,~g of phytohemagglutinin are bound per lymphocyte. This finding correlates well with the observation that lymphocytes from chronic lymphocytic leukemic patients with high peripheral lymphocyte counts do not respond to phytohemagglutinin as well as do lymphocytes from chronic lymphocytic leukemic patients with low lymphocyte counts33 4.
LYMPHOCYTE
COUNT(fburondr~31
Fig. 2. Relation of phytohemagglutinin lymphocyte count. 0, normal subjects;
(PHA) binding to lymphocytes to the peripheral blood 0, patients with chronic lymphocytic leukemia.
We have recently demonstrated that the erythroagglutinating phytohemagglutinin binds to a glycoprotein structure on the erythrocyte and lymphocyte cell surfacell. The specificity for binding was found to reside primarily in the galactose moiety of an oligosaccharide chain having the terminal sequence of sialic acid-f galactose --f K. If there is one sialic acid residue per phytohemagglutinin receptor site, then approx. 2.7.10~ molecules of sialic acid on the lymphocyte cell surface are involved in phytohemagglutinin binding. This figure represents about I o/0 of the total sialic acid molecules on the normal lymphocyte cell surface (3.1.10s molecules/cell)* . If the reduced number of phytohemagglutinin
receptor sites in chronic lymphocytic
leukemic
lymphocytes represented a selective loss of these receptor sites or a masking of these sites due to an alteration in the conformation of the cell surfacelz, the total cell surface sialic acid content of chronic lymphocytic leukemic lymphocytes would be expected to be unchanged. However, if the loss of receptor sites was but one manifestation of a grossly altered cell surface, then the total sialic acid content of chronic lymphocytic leukemic lymphocytes might differ from the normal. When the sialic acid content of lymphocytes was measured in these two groups, it was found that chronic lymphocytic leukemic lymphocytes contain much less sialic acid than do normal lymphocytes. The mean value (& S.E.) of total sialic acid per I* IO* lymphocytes for 8 normal subjects was 52 f 4 nmoles and for 12 chronic lymphocytic leukemic patients, 29 + 3 nmoles. This difference is highly significant (P < 0.001). When either normal or chronic lymphocytic leukemic lymphocytes were treated with the enzyme neuraminidase, about 60 0/Oof the total cell sialic acid was released. Since the enzyme only has access to sialic acid residues on the cell surface, the chronic lymphocytic leukemic lympho* This value is based on our finding that there are approx. 52 nmoles of sialic acid per I. normal lymphocytes. Biochim.
Biophys.
Acta,
192 (1969) 542-545
IO*
545
SHORT COMMUNICATIONS
cytes also have less sialic acid on their surfaces than do normal lymphocytes although the cellular distribution on a percentage basis is the same. These experiments demonstrate that the cell surface of chronic lymphocytic leukemic lymphocytes differs considerably from the cell surface of normal lymphocytes. The decreased number of phytohemagglutinin receptor sites is probably just one reflection of the altered cell surface. The findir@ that the ABH blood group antigens have markedly decreased or even absent reactivity in chronic lymphocytic leukemic lymphocytes compared to normal lymphocytes is consistent with this proposal. This research was supported in part by grants from the U.S. Public Health Service (HE-ooozz and ROI CA-08759) and the American Cancer Society (PRA31). Washingtovz
University
Sch,ool of Medicine,
STUART
KOKNFELD
St. Louis, MO. 63 IIO (U.S.A.) I P. C. NOWELL, Exptl. Cell Res., rg (1960) 267. 2 J. H. ROBBINS, Science, 146 (1964) 1648. 3 R. SCHREK, Arch. Pathol., 83 (1967) 58. 4 K. HAVEMANN AND A. D. RUBIN, Proc. Sot. Expt. Biol., 127 (1968) 668. 5 T. WEBER, C.T. NORDMAN AND R. GRKsBECK,S~~~~. J. Haematol., 4 (1967) 77. 6 II. _\WAI AND E. B. BROWN, J. Lab. Clin. Med., 61 (1963) 363. 7 H. EAGLE, Science, 130 (1959) 432. 8 bl. TUNIS, Federation Proc., 27 (1968) 813. 9 L. WARREN, J. Biol. Chem., 234 (1959) 1971.
IO II 12 13
T. L. STECK AND D. F. H. WALLACH,Biochim.Biophys.Acta.g7 (1965) 510. S. KORNFELD AND R. KORNFELD, Proc. Natl. Acad. Sci U S., in the press. S. HAKAMORI, C. TEATHER AND H. ANDREWS, Biochem. Biophys. Res. Commun., J. I.BRODY AND L. H. BEIZER, J. C&n. Invest., 44 (1965) 1582.
33 (1968) 563.
Received September rst, 1969 Biochim.
Biophys.
Acta,
Igz (1969) 542-545
BBA 23544 Properties and developmental
changes of human hepatic aryl pyruvate
enzymes
The development and properties of liver tyrosine aminotransferase (L-tyrosine : z-oxoglutarate aminotransferase, EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (p-hydroxyphenylpyruvate, ascorbate : 0, oxidoreductase (hydroxylating), EC 1.14.2.2) have been studied extensively in animals1-3 and occasionally in man4. Here, changes in tyrosine aminotransferase, p-hydroxyphenylpyruvate hydroxylase and phenylpyruvate tautomerase (phenylpyruvate keto-enol-isomerase, EC 5.x.2.1), related to maturation of the human liver, are reported. These enzymes are similarily distributed (active in the liver, kidneys, heart and spleen; absent from the blood and muscles5y6)and have centers specifically binding p-hydroxyphenylpyruvate and phenylpyruvate7-g. Liver specimens were taken at autopsy within 12 h after death (from brain damage, asphyxia, heart arrest or route accident). Part of each sample was promptly Biochim.
Biophys.
Acta,
192 (1969) 545-548
542 BBA 23 552 Decreased phytohemagglutinin
receptor
sites in chronic lymphocytic
leukemia When lymphocytes from normal human subjects are incubated with phytohemagglutinin, they transform into large cells capable of undergoing mitosis. In contrast, lymphocytes from patients with chronic lymphocytic leukemia show both a delayed and an impaired blastogenic response to phytohemagglutininl-4. The mechanism for this defective responsiveness has not been elucidated. Since the binding of phytohemagglutinin to cell surface receptor sites appears to be the initial step in the sequence of events leading to mitogenesis, we have examined normal and chronic lymphocytic leukemic lymphocytes in order to determine whether a defect in chronic lymphocytic leukemic lymphocytes could be localized at this step. Highly purified phytohemagglutinin was prepared from Bacto-Phytohemagglutinin-P (Difco Laboratories) by SE-Sephadex chromatography as described by WEBER et aL5. The material in Peak III (ref. 5) was further purified by gel filtration on Sephadex G-150 to give a protein with potent erythroagglutinating as well as lymphocyte stimulating properties. This protein, after being iodinated by the method of AWAI AND BROWNS, retained its full erythroagglutinating activity. Lymphocytes were isolated as follows: For every 85 ml of fresh heparinized blood 15 ml of Gy,6 dextran in saline were added and the red cells were allowed to settle at 37’. The supernatant fluid was freed of granulocytes by passage through a column of nylon fibers packed into a 5o-ml glass syringe. The filtrate was sedimented twice at 400 x g for 7 min, cold distilled water was added to the cell pellet to osmotically lyse the remaining red blood cells, and after 25 set, enough 3.474 saline was added to restore isotonicity, and the suspension was centrifuged for 7 min at 400 x g. The white cell pellet containing go--100% lymphocytes was suspended in Eagle’s minimal essential media7 at a concentration of about 20.10~ cells per cm3. The final cell suspensions contained platelet per 2 lymphocytes.
less than I red cell per IOO lymphocytes and I or fewer The binding of phytohemagglutinin to lymphocytes was
not affected by the osmotic shock step. Total cellular sialic acid was determined by heating intact cells at 100’ for 1.5 min in I M HCl (ref. 8). The cellular debris was removed by centrifugation and the released sialic acid was measured by the method of WARRENS. Enzyme releasable sialic acid was determined in the same way after lymphocytes were incubated in minimal essential medium containing 50 units/ml of neuraminidase (Calbiochem Corp.) for I h at 37’. The binding reactions were carried out in plastic counting tubes in minimal essential medium which contained in 0.4 ml : from 1.25 to 12.5 pg [1311]phytohemagglutinin (specific activity 6.25.10~ counts/min per pg), 2 mg bovine serum albumin, and 2.106 lymphocytes. After 30 min incubation at room temperature, the reaction had come to equilibrium and 6 ml of saline were added, the cells were sedimented, washed with another 6 ml saline, and then the bound radioactivity was determined in a Packard Autogamma counter. Appropriate corrections were made for nonspecific binding to the plastic tubes (in the absence of cells) which accounted for less than 5 y0 of the total counts bound. The data were plotted as described by STECK AND Biochim.
Biophys.
Acta,
192 (1969) 542-545
SHORT COMMUNICATIONS
543
The amount of [1311]phvtollemagglutinin bound was directly proportional ” to the number of lymphocytes present. When the ability of normal and chronic lymphocytic leukemic lymphocytes to
WALLACH~.
bind [1311]pllytohemagglutinin was measured as a function of [1311]phytohemagglutinin concentration, it was found that chronic lymphocytic leukemic lymphocytes bound significantly less phytohemagglutinin than did normal lymphocytes. Typical binding curves are shown in Fig. I. In all three instances the apparent dissociation constant (K) for phytohemagglutinin bindingwas approximately the same (8.4--10.4,~g phytohemagglutinin per ml), while the maximal amount of phytohemagglutinin bound per 2.10~ lymphocytes differed considerably (1.48 pg by the normal lymphocytes compared to 0.36 and 0.80 ,ug by the chronic lymphocytic leukemic lymphocytes. Lymphocytes from 12 normal donors had a mean K (* S.E.) of 12.0 f 1.1 peg phytohemagglutinin per ml while lymphocytes from 14 patients with chronic lymphocytic leukemia had a mean K of 10.3 f 0.7. These values are not significantly different (P > 0.1). In contrast, the lymphocytes from the 12 normal donors bound on the average 1.16 f 0.08 ,ug phytohemagglutinin per ~2.10~ cells while the chronic lymphocytic leukemic lymphocytes (14 donors) bound only 0.49 f 0.06 pg phytohemagglutinin per 2. IO6 cells, an amount significantly less than the normal (P < 0.001). If the molecular weight of the phytohemagglutinin is assumed to be 128000 (ref. IO), one can calculate that the average number of receptor sites per normal lymphocyte is 2.7.10~ and per chronic lymphocytic leukemic lymphocyte, 1.15.10~. This estimate compares with 3.4.10~ receptor sites per erythrocyteii and 6.6.107 receptor sites per Ehrlich ascites carcinoma celllo. The amount
of phytohemagglutinin
bound per chronic
lymphocytic
leukemic
I.2
Fig. I. The binding of phytohemagglutinin (PHA) to lymphocytes. Normal and chronic lymphocytic leukemic lymphocytes were incubated with [1311]phytohemagglutinin as described in the text. The data have been plotted (B) by the method of STECK AND WALLACH~’ according to the equation : C [PHA
bound] = &
’ [PiA]
+ i
where [PHA] = concentration free phytohemagglutinin (,@. n = number of PHA binding sites per cell expressed as pg phytohemagglutinin bound per lymphocyte, and C = number of cells. The dissociation constant K for maximal phytohemagglutinin binding to lymphocytes was determined from the I/[PHA] intercept and the pg phytohemagglutinin bound per lymphocyte, n, from the C/[PHA bound] intercept. o-0, normal donor; o-0, chronic lymphocytic leukemic donor with a peripheral lymphocyte count of r4 roo/mms; A-A, chronic lymphocytic leukemic donor with a lymphocyte count of 54 5oo/mm3. Biochim.
Bioflhys. Acta, I92 (1969) 542-545
544
SHORT COMMUNICATIONS
lymphocyte is inversely related to the patient’s peripheral lymphocyte count (Fig. 2) ; thus, the higher the patient’s lymphocyte count the fewer ,~g of phytohemagglutinin are bound per lymphocyte. This finding correlates well with the observation that lymphocytes from chronic lymphocytic leukemic patients with high peripheral lymphocyte counts do not respond to phytohemagglutinin as well as do lymphocytes from chronic lymphocytic leukemic patients with low lymphocyte counts33 4.
LYMPHOCYTE
COUNT(fburondr~31
Fig. 2. Relation of phytohemagglutinin lymphocyte count. 0, normal subjects;
(PHA) binding to lymphocytes to the peripheral blood 0, patients with chronic lymphocytic leukemia.
We have recently demonstrated that the erythroagglutinating phytohemagglutinin binds to a glycoprotein structure on the erythrocyte and lymphocyte cell surfacell. The specificity for binding was found to reside primarily in the galactose moiety of an oligosaccharide chain having the terminal sequence of sialic acid-f galactose --f K. If there is one sialic acid residue per phytohemagglutinin receptor site, then approx. 2.7.10~ molecules of sialic acid on the lymphocyte cell surface are involved in phytohemagglutinin binding. This figure represents about I o/0 of the total sialic acid molecules on the normal lymphocyte cell surface (3.1.10s molecules/cell)* . If the reduced number of phytohemagglutinin
receptor sites in chronic lymphocytic
leukemic
lymphocytes represented a selective loss of these receptor sites or a masking of these sites due to an alteration in the conformation of the cell surfacelz, the total cell surface sialic acid content of chronic lymphocytic leukemic lymphocytes would be expected to be unchanged. However, if the loss of receptor sites was but one manifestation of a grossly altered cell surface, then the total sialic acid content of chronic lymphocytic leukemic lymphocytes might differ from the normal. When the sialic acid content of lymphocytes was measured in these two groups, it was found that chronic lymphocytic leukemic lymphocytes contain much less sialic acid than do normal lymphocytes. The mean value (& S.E.) of total sialic acid per I* IO* lymphocytes for 8 normal subjects was 52 f 4 nmoles and for 12 chronic lymphocytic leukemic patients, 29 + 3 nmoles. This difference is highly significant (P < 0.001). When either normal or chronic lymphocytic leukemic lymphocytes were treated with the enzyme neuraminidase, about 60 0/Oof the total cell sialic acid was released. Since the enzyme only has access to sialic acid residues on the cell surface, the chronic lymphocytic leukemic lympho* This value is based on our finding that there are approx. 52 nmoles of sialic acid per I. normal lymphocytes. Biochim.
Biophys.
Acta,
192 (1969) 542-545
IO*
545
SHORT COMMUNICATIONS
cytes also have less sialic acid on their surfaces than do normal lymphocytes although the cellular distribution on a percentage basis is the same. These experiments demonstrate that the cell surface of chronic lymphocytic leukemic lymphocytes differs considerably from the cell surface of normal lymphocytes. The decreased number of phytohemagglutinin receptor sites is probably just one reflection of the altered cell surface. The findir@ that the ABH blood group antigens have markedly decreased or even absent reactivity in chronic lymphocytic leukemic lymphocytes compared to normal lymphocytes is consistent with this proposal. This research was supported in part by grants from the U.S. Public Health Service (HE-ooozz and ROI CA-08759) and the American Cancer Society (PRA31). Washingtovz
University
Sch,ool of Medicine,
STUART
KOKNFELD
St. Louis, MO. 63 IIO (U.S.A.) I P. C. NOWELL, Exptl. Cell Res., rg (1960) 267. 2 J. H. ROBBINS, Science, 146 (1964) 1648. 3 R. SCHREK, Arch. Pathol., 83 (1967) 58. 4 K. HAVEMANN AND A. D. RUBIN, Proc. Sot. Expt. Biol., 127 (1968) 668. 5 T. WEBER, C.T. NORDMAN AND R. GRKsBECK,S~~~~. J. Haematol., 4 (1967) 77. 6 II. _\WAI AND E. B. BROWN, J. Lab. Clin. Med., 61 (1963) 363. 7 H. EAGLE, Science, 130 (1959) 432. 8 bl. TUNIS, Federation Proc., 27 (1968) 813. 9 L. WARREN, J. Biol. Chem., 234 (1959) 1971.
IO II 12 13
T. L. STECK AND D. F. H. WALLACH,Biochim.Biophys.Acta.g7 (1965) 510. S. KORNFELD AND R. KORNFELD, Proc. Natl. Acad. Sci U S., in the press. S. HAKAMORI, C. TEATHER AND H. ANDREWS, Biochem. Biophys. Res. Commun., J. I.BRODY AND L. H. BEIZER, J. C&n. Invest., 44 (1965) 1582.
33 (1968) 563.
Received September rst, 1969 Biochim.
Biophys.
Acta,
Igz (1969) 542-545
BBA 23544 Properties and developmental
changes of human hepatic aryl pyruvate
enzymes
The development and properties of liver tyrosine aminotransferase (L-tyrosine : z-oxoglutarate aminotransferase, EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (p-hydroxyphenylpyruvate, ascorbate : 0, oxidoreductase (hydroxylating), EC 1.14.2.2) have been studied extensively in animals1-3 and occasionally in man4. Here, changes in tyrosine aminotransferase, p-hydroxyphenylpyruvate hydroxylase and phenylpyruvate tautomerase (phenylpyruvate keto-enol-isomerase, EC 5.x.2.1), related to maturation of the human liver, are reported. These enzymes are similarily distributed (active in the liver, kidneys, heart and spleen; absent from the blood and muscles5y6)and have centers specifically binding p-hydroxyphenylpyruvate and phenylpyruvate7-g. Liver specimens were taken at autopsy within 12 h after death (from brain damage, asphyxia, heart arrest or route accident). Part of each sample was promptly Biochim.
Biophys.
Acta,
192 (1969) 545-548