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Ribosome-granular material complexes in human leukemic lymphoblasts exposed to vinblastine sulfate
© 1970 by Academic _Press, Inc.
272
J. ULTRASTRUCTURE RESEARCH 31, 272-281 (1970)
Ribosome-Granular Material Complexes in Human Leukemic Lymphoblasts Exposed to Vinblastine Sulfate AWTAR KRISHAN
Children's Cancer Research Foundation and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 Received August 4, 1969
Human leukemic lymphoblasts exposed to Velban show prominent proteinaceous crystals and large complexes of ribosomes (which are arranged in clusters and helices) in association with fine granular, electron dense material. This granular material shows relatively greater electron density than the crystals and lacks the prominent linearity of the crystals. In some cross sections tubular profiles are seen interspersed between the granular material and the ribosomes. It is suggested that the ribosomes are involved in the synthesis of the electrondense material which is subsequently organized into the prominent crystals.
The presence of large concentric whorls of granular endoplasmic reticulum, annulate lamellae (6), proteinaceous crystals (2, 9), and the unusual abundance of aggregates of free ribosomes and polyribosomes (2, 7, 9) in cells exposed to Velban (VLB) and vincristine sulfate (VCR), both in vivo and in vitro, suggest that these alkaloids may stimulate synthesis of some cellular proteins. In cells reincubated in fresh medium after exposure to VCR, bundles of 50-80 A filaments are seen attached to the crystals. Incubation for longer periods of time in fresh medium shows the gradual replacement of the crystals by wavy patches of filamentous material. These observations suggest that the VCR-induced crystals and the filaments which replace them in reincubated cells probably share a common pool of cellular proteins and are interchangeable (7). In Earle's L-929 cells exposed to VCR, large rows of ribosomes arranged in clusters and in helical polyribosome-like configurations are often associated with the crystals and in many sections filaments extending from individual helical polyribosomes into the crystals are seen (7). In an earlier study, association of ribosomes and polyribosomes with what were described as "abnormal large microtubules" was reported in cells exposed to VLB and VCR (2). In a recent study we reported in VCR-treated L-cells the association of ribosomes and helical polyribosomes with "stalks" of fine,
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moderately electron dense material (7). As seen in the present study, human leukemic lymphoblasts (CCRF-CEM) exposed to VCR or VLB, show unusually large aggregates of ribosomes in association with fine, granular electron dense material. Observations from the present study suggest that these long rows of ribosomes are involved in the synthesis of the associated electron dense material which may be the precursor material for the crystals. MATERIALS AND METHODS Human leukemic lymphoblasts (CCRF-CEM) (4) from suspension cultures were treated with 10/~g/ml of vinblastine sulfate (Velban, E. Lilly, Indianapolis) for 3 hours. Cell buttons retrieved after centrifugation were fixed in 2 % glutaraldehyde at room temperature for 15 minutes, washed in 6 % buffered sucrose, and postfixed in 2 % osmium tetroxide. Dehydration in graded acetone series was followed by embedding in an Epon-Araldite mixture. Thin sections were stained with lead citrate solution and examined in a Philips 300 electron microscope. OBSERVATIONS Crystals induced by VLB or VCR in human leukemic lymphoblasts (CCRFCEM) are morphologically similar to those described earlier in Earle's L-929 fibroblasts, platelets, human neurons and rabbit neurons, and oligodendroglia (2, 8, 9-11). Figure 1 shows a group of crystals in the pericentriolar area of a lymphoblast. In longitudinal sections these crystals show a substructure of parallel filaments 250280 A apart (Fig. 1) whereas in cross sections (Fig. 2) a honeycomb-like substructure of circular subunits is seen. Arrows point to small electron dense granules in the center of some of the circular cross sections. These granules (80-90 A) are slightly smaller in diameter than the ribosomes (120-125 A) in the surrounding cytoplasm. The CCRFCEM lymphoblasts exposed to Velban or vincristine for 3 hours have prominent crystals and unusually large complexes of granules and electron dense material in the cytoplasm. In Fig. 3, arrows point to a 2.5 # long complex in the cytoplasm of a lymphoblast. Fig. 4 shows at a higher magnification the pericentriolar area of a lymphoblast with crystals (CR) and a prominent complex of electron dense material and granules (arrows), the latter being similar in size and electron density to the surrounding cytoplasmic ribosomes. The ribosome-associated, electron dense material shows a faint linear arrangement and greater electron density than the crystals. Figure 5 shows at a higher magnification the association of the ribosomes and the electron dense material. In these photomicrographs, ribosomes are interspersed in small clumps between the rows of the electron dense granular material. Although fine linearity of the electron dense material is evident, no apparent resemblance to the "abnormal large
274
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FIG. 3. Arrows point to a large complex of granules and electron dense material in the cytoplasm, x 15,500.
m i c r o t u b u l e s " r e p o r t e d earlier (2) o r to the p r o m i n e n t linear a r r a n g e m e n t of the crystals can be recognized at this stage. In Fig. 6, 18-20 linear rows of ribosomes, m a n y of which a p p e a r to be a r r a n g e d in long helices are seen interspersed between the electron dense material. I n some of the l o n g e r helices, 15 o r m o r e i n d i v i d u a l r i b o s o m e s can be recognized. F i g u r e 7 shows a transverse section t h r o u g h one of these complexes. The g r a n u l a r electron dense m a t e r i a l is seen a r o u n d small clusters of r i b o s o m e s (arrows). I n occasional cross sections of the electron dense m a t e r i a l a n d r i b o s o m e complexes, t u b u l a r cross sections interspersed with r i b o s o m e s a n d the dense m a t e r i a l can be seen (Fig. 8). A t this stage, these complexes resemble those described earlier b y Bensch a n d M a l a w i s t a (2). I n All figures are from CCRF-CEM culture incubated with Velban for 3 hours. Magnification markers on all figures indicate 1 micrometer. FIG. 1. A group of Velban-induced crystals sectioned longitudinally in the pericentriolar area of a lymphoblast. × 26,700. FIG. 2. A crystal in cross section. Arrows point to small granules in the center of some of the circular subunits in this honeycomb-like structure. × 77,400.
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these profiles relatively smaller numbers of associated ribosomes are seen compared to the cross sections that show the granular material without any tubular organization. In Fig. 9, ribosomes and helical polyribosomes are seen in close association with electron dense material, which in turn seems to be in continuity with a crystal. Arrow points to a few remaining ribosomes, which are seen between the linear filaments of the crystal. DISCUSSION Earlier evidence from histochemical studies and enzyme extractions (7) has clearly suggested that the VLB- and VCR-induced crystals are proteinaceous and do not contain appreciable amounts of either R N A or DNA. Incubation of cultures with puromycin, actinomycin D, p-fluorophenylalanine (2, 7), or cycloheximide (8) before and/or during the exposure to VCR or VLB has failed to prevent the crystal formation. Preliminary evidence from leucine-aH labeling experiments (8) also suggests that a major part of the crystals are assembled from preformed proteins, possibly the precursors of the microtubular-filamentous elements. Ribosome-associated complexes described variously as granule-lamellae or polysome-lamellae complexes, which are more or less similar to those described in the present study, have been reported recently in plant cells (1), in cells from the proximal renal tubules of a monkey (3), and in adenoma cells from a human adrenal cortex (5). While the ribosome-lamellae complexes in the monkey renal tubular cells cannot be related to drug administration, it is not known whether the material from the human adrenal cortex was obtained from a patient who was being treated with drugs. The presence of large amounts of ribosomes and helical polyribosomes in association with crystals and with the masses of electron dense material, as seen in the present study, suggests that in addition to arresting cells in mitosis and causing the formation of crystals from preformed cellular proteins, VLB and VCR may stimulate the further production of protein subunits, the probable precursors of the crystals (2). As shown in the present study, in many sections the electron dense material associated with the ribosomes shows a faint linearity while in some other sections it shows reduced electron density and the presence of tubular profiles identical in diameter to these seen in the cross sections of the crystals. Based on these observations, it is reasonable to speculate that the following events occur in cells exposed to vinca alkaloids VLB and VCR in concentrations used in the present study.
Fla. 4. Two crystals (CR) and a complex of granules and electron dense material (arrow) at a higher magnification. The granules resemble the surrounding cytoplasmic ribosomes in electron density and size. × 29,500. FI~. 5. The association between the ribosomes and the electron dense material, x 102,900.
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Fro. 6. Linear, helical arrangements of ribosomes interspersed between the electron dense material. x 68,800.
FIo. 7. A transverse section through a complex. Arrows point to small clusters of ribosomes surrounded by electron dense material, x 91,000. FIo. 8. Occasionally seen tubular cross sections interspersed among ribosomes and the electron dense material, x 86,200.
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Fro. 9. Ribosomes and helical polyribosomes in close association with the electron dense material, which in turn is associated with a crystal. Arrow points to ribosomes between the linear filaments of the crystal, x 62,500.
1. Initial breakdown of cellular microtubles and reformation of their constituent and/or precursor proteins into crystals showing a linear arrangement of filaments in longitudinal sections and tubular profiles in cross sections. 2. Stimulation of further synthesis of these precursor proteins through the activity of the ribosomes and helical polyribosomes. The electron dense material seen in association with the ribosomes which shows a faint linearity but is much more electrondense than the constituent material of the crystals, could be this newly synthesized precursor protein. 3. Reorganization of this electron dense material into tubular profiles followed by elimination of the ribosomes and the formation of the crystalline structures. Further studies involving the use of protein synthesis inhibitors and all-labeled amino acids are in progress to test the validity of the hypothesis proposed above. These studies were supported in part by research grants C-6516 from the National Cancer Institute, and FR-05526 from the Division of Research Facilities and Resources, National Institutes of Health; the legacy of Loula D. Lasker; the Albert and Mary Lasker Founda-
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tion, New York; and the Alvin T. and Viola D. Fuller Cancer Research Unit Grant, American Cancer Society (Massachusetts Division), Inc. We are grateful to Drs. L. Desai, H. Lazarus and G. E. Foley for the cultures of CCRFCEM cells and to Drl Betty G. Uzman for her critical reading of the manuscript. The technical help of Mrs. E. Galvanek and Miss C. Calabia and the secretarial assistance of Miss E. Monkouski is gratefully acknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11.
BARTELS,P. and WEIER,T. E., J. Cell Biol. 33, 243 (1967). BENSCH, K. G. and MALAWISTA,S. E., 3". Cell Biol. 40, 95 (1969). BULGER, R. E., J. Ultrastruct. Res. 24, 150 (1968). FOLEY, G. E., LAZARUS,H., FARBER, S., UZMAN, B. G., BOONE, B. A., and MCCARTHY, R. E., Cancer Res. 18, 522 (1965). HOSmNO, M., J. Ultrastruct. Res. 27, 205 (1969). KRISHAN,A., HSU, D. and HuTcmNS, P., J. Cell Biol. 39, 211 (1968). KRISHAN,A. and Hsu, D., J. Ceil BioL 43, 553 (1969). - - - - unpublished observations. SCI~OCHET,S. S., JR., LAMPERT,P. W. and EARLE, K. M., J. Neuropathol. Exptl. Neurol. 27, 645 (1968). -Exptl. Neurol. 23, 113 (1969). WroTE, J. G., Am. J. Pathol. 53, 447 (1968).
272
J. ULTRASTRUCTURE RESEARCH 31, 272-281 (1970)
Ribosome-Granular Material Complexes in Human Leukemic Lymphoblasts Exposed to Vinblastine Sulfate AWTAR KRISHAN
Children's Cancer Research Foundation and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 Received August 4, 1969
Human leukemic lymphoblasts exposed to Velban show prominent proteinaceous crystals and large complexes of ribosomes (which are arranged in clusters and helices) in association with fine granular, electron dense material. This granular material shows relatively greater electron density than the crystals and lacks the prominent linearity of the crystals. In some cross sections tubular profiles are seen interspersed between the granular material and the ribosomes. It is suggested that the ribosomes are involved in the synthesis of the electrondense material which is subsequently organized into the prominent crystals.
The presence of large concentric whorls of granular endoplasmic reticulum, annulate lamellae (6), proteinaceous crystals (2, 9), and the unusual abundance of aggregates of free ribosomes and polyribosomes (2, 7, 9) in cells exposed to Velban (VLB) and vincristine sulfate (VCR), both in vivo and in vitro, suggest that these alkaloids may stimulate synthesis of some cellular proteins. In cells reincubated in fresh medium after exposure to VCR, bundles of 50-80 A filaments are seen attached to the crystals. Incubation for longer periods of time in fresh medium shows the gradual replacement of the crystals by wavy patches of filamentous material. These observations suggest that the VCR-induced crystals and the filaments which replace them in reincubated cells probably share a common pool of cellular proteins and are interchangeable (7). In Earle's L-929 cells exposed to VCR, large rows of ribosomes arranged in clusters and in helical polyribosome-like configurations are often associated with the crystals and in many sections filaments extending from individual helical polyribosomes into the crystals are seen (7). In an earlier study, association of ribosomes and polyribosomes with what were described as "abnormal large microtubules" was reported in cells exposed to VLB and VCR (2). In a recent study we reported in VCR-treated L-cells the association of ribosomes and helical polyribosomes with "stalks" of fine,
RIBOSOME COMPLEXES
273
moderately electron dense material (7). As seen in the present study, human leukemic lymphoblasts (CCRF-CEM) exposed to VCR or VLB, show unusually large aggregates of ribosomes in association with fine, granular electron dense material. Observations from the present study suggest that these long rows of ribosomes are involved in the synthesis of the associated electron dense material which may be the precursor material for the crystals. MATERIALS AND METHODS Human leukemic lymphoblasts (CCRF-CEM) (4) from suspension cultures were treated with 10/~g/ml of vinblastine sulfate (Velban, E. Lilly, Indianapolis) for 3 hours. Cell buttons retrieved after centrifugation were fixed in 2 % glutaraldehyde at room temperature for 15 minutes, washed in 6 % buffered sucrose, and postfixed in 2 % osmium tetroxide. Dehydration in graded acetone series was followed by embedding in an Epon-Araldite mixture. Thin sections were stained with lead citrate solution and examined in a Philips 300 electron microscope. OBSERVATIONS Crystals induced by VLB or VCR in human leukemic lymphoblasts (CCRFCEM) are morphologically similar to those described earlier in Earle's L-929 fibroblasts, platelets, human neurons and rabbit neurons, and oligodendroglia (2, 8, 9-11). Figure 1 shows a group of crystals in the pericentriolar area of a lymphoblast. In longitudinal sections these crystals show a substructure of parallel filaments 250280 A apart (Fig. 1) whereas in cross sections (Fig. 2) a honeycomb-like substructure of circular subunits is seen. Arrows point to small electron dense granules in the center of some of the circular cross sections. These granules (80-90 A) are slightly smaller in diameter than the ribosomes (120-125 A) in the surrounding cytoplasm. The CCRFCEM lymphoblasts exposed to Velban or vincristine for 3 hours have prominent crystals and unusually large complexes of granules and electron dense material in the cytoplasm. In Fig. 3, arrows point to a 2.5 # long complex in the cytoplasm of a lymphoblast. Fig. 4 shows at a higher magnification the pericentriolar area of a lymphoblast with crystals (CR) and a prominent complex of electron dense material and granules (arrows), the latter being similar in size and electron density to the surrounding cytoplasmic ribosomes. The ribosome-associated, electron dense material shows a faint linear arrangement and greater electron density than the crystals. Figure 5 shows at a higher magnification the association of the ribosomes and the electron dense material. In these photomicrographs, ribosomes are interspersed in small clumps between the rows of the electron dense granular material. Although fine linearity of the electron dense material is evident, no apparent resemblance to the "abnormal large
274
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275
FIG. 3. Arrows point to a large complex of granules and electron dense material in the cytoplasm, x 15,500.
m i c r o t u b u l e s " r e p o r t e d earlier (2) o r to the p r o m i n e n t linear a r r a n g e m e n t of the crystals can be recognized at this stage. In Fig. 6, 18-20 linear rows of ribosomes, m a n y of which a p p e a r to be a r r a n g e d in long helices are seen interspersed between the electron dense material. I n some of the l o n g e r helices, 15 o r m o r e i n d i v i d u a l r i b o s o m e s can be recognized. F i g u r e 7 shows a transverse section t h r o u g h one of these complexes. The g r a n u l a r electron dense m a t e r i a l is seen a r o u n d small clusters of r i b o s o m e s (arrows). I n occasional cross sections of the electron dense m a t e r i a l a n d r i b o s o m e complexes, t u b u l a r cross sections interspersed with r i b o s o m e s a n d the dense m a t e r i a l can be seen (Fig. 8). A t this stage, these complexes resemble those described earlier b y Bensch a n d M a l a w i s t a (2). I n All figures are from CCRF-CEM culture incubated with Velban for 3 hours. Magnification markers on all figures indicate 1 micrometer. FIG. 1. A group of Velban-induced crystals sectioned longitudinally in the pericentriolar area of a lymphoblast. × 26,700. FIG. 2. A crystal in cross section. Arrows point to small granules in the center of some of the circular subunits in this honeycomb-like structure. × 77,400.
276
KRISHAN
these profiles relatively smaller numbers of associated ribosomes are seen compared to the cross sections that show the granular material without any tubular organization. In Fig. 9, ribosomes and helical polyribosomes are seen in close association with electron dense material, which in turn seems to be in continuity with a crystal. Arrow points to a few remaining ribosomes, which are seen between the linear filaments of the crystal. DISCUSSION Earlier evidence from histochemical studies and enzyme extractions (7) has clearly suggested that the VLB- and VCR-induced crystals are proteinaceous and do not contain appreciable amounts of either R N A or DNA. Incubation of cultures with puromycin, actinomycin D, p-fluorophenylalanine (2, 7), or cycloheximide (8) before and/or during the exposure to VCR or VLB has failed to prevent the crystal formation. Preliminary evidence from leucine-aH labeling experiments (8) also suggests that a major part of the crystals are assembled from preformed proteins, possibly the precursors of the microtubular-filamentous elements. Ribosome-associated complexes described variously as granule-lamellae or polysome-lamellae complexes, which are more or less similar to those described in the present study, have been reported recently in plant cells (1), in cells from the proximal renal tubules of a monkey (3), and in adenoma cells from a human adrenal cortex (5). While the ribosome-lamellae complexes in the monkey renal tubular cells cannot be related to drug administration, it is not known whether the material from the human adrenal cortex was obtained from a patient who was being treated with drugs. The presence of large amounts of ribosomes and helical polyribosomes in association with crystals and with the masses of electron dense material, as seen in the present study, suggests that in addition to arresting cells in mitosis and causing the formation of crystals from preformed cellular proteins, VLB and VCR may stimulate the further production of protein subunits, the probable precursors of the crystals (2). As shown in the present study, in many sections the electron dense material associated with the ribosomes shows a faint linearity while in some other sections it shows reduced electron density and the presence of tubular profiles identical in diameter to these seen in the cross sections of the crystals. Based on these observations, it is reasonable to speculate that the following events occur in cells exposed to vinca alkaloids VLB and VCR in concentrations used in the present study.
Fla. 4. Two crystals (CR) and a complex of granules and electron dense material (arrow) at a higher magnification. The granules resemble the surrounding cytoplasmic ribosomes in electron density and size. × 29,500. FI~. 5. The association between the ribosomes and the electron dense material, x 102,900.
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277
Fro. 6. Linear, helical arrangements of ribosomes interspersed between the electron dense material. x 68,800.
FIo. 7. A transverse section through a complex. Arrows point to small clusters of ribosomes surrounded by electron dense material, x 91,000. FIo. 8. Occasionally seen tubular cross sections interspersed among ribosomes and the electron dense material, x 86,200.
280
KRISHAN
Fro. 9. Ribosomes and helical polyribosomes in close association with the electron dense material, which in turn is associated with a crystal. Arrow points to ribosomes between the linear filaments of the crystal, x 62,500.
1. Initial breakdown of cellular microtubles and reformation of their constituent and/or precursor proteins into crystals showing a linear arrangement of filaments in longitudinal sections and tubular profiles in cross sections. 2. Stimulation of further synthesis of these precursor proteins through the activity of the ribosomes and helical polyribosomes. The electron dense material seen in association with the ribosomes which shows a faint linearity but is much more electrondense than the constituent material of the crystals, could be this newly synthesized precursor protein. 3. Reorganization of this electron dense material into tubular profiles followed by elimination of the ribosomes and the formation of the crystalline structures. Further studies involving the use of protein synthesis inhibitors and all-labeled amino acids are in progress to test the validity of the hypothesis proposed above. These studies were supported in part by research grants C-6516 from the National Cancer Institute, and FR-05526 from the Division of Research Facilities and Resources, National Institutes of Health; the legacy of Loula D. Lasker; the Albert and Mary Lasker Founda-
RIBOSOME COMPLEXES
281
tion, New York; and the Alvin T. and Viola D. Fuller Cancer Research Unit Grant, American Cancer Society (Massachusetts Division), Inc. We are grateful to Drs. L. Desai, H. Lazarus and G. E. Foley for the cultures of CCRFCEM cells and to Drl Betty G. Uzman for her critical reading of the manuscript. The technical help of Mrs. E. Galvanek and Miss C. Calabia and the secretarial assistance of Miss E. Monkouski is gratefully acknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11.
BARTELS,P. and WEIER,T. E., J. Cell Biol. 33, 243 (1967). BENSCH, K. G. and MALAWISTA,S. E., 3". Cell Biol. 40, 95 (1969). BULGER, R. E., J. Ultrastruct. Res. 24, 150 (1968). FOLEY, G. E., LAZARUS,H., FARBER, S., UZMAN, B. G., BOONE, B. A., and MCCARTHY, R. E., Cancer Res. 18, 522 (1965). HOSmNO, M., J. Ultrastruct. Res. 27, 205 (1969). KRISHAN,A., HSU, D. and HuTcmNS, P., J. Cell Biol. 39, 211 (1968). KRISHAN,A. and Hsu, D., J. Ceil BioL 43, 553 (1969). - - - - unpublished observations. SCI~OCHET,S. S., JR., LAMPERT,P. W. and EARLE, K. M., J. Neuropathol. Exptl. Neurol. 27, 645 (1968). -Exptl. Neurol. 23, 113 (1969). WroTE, J. G., Am. J. Pathol. 53, 447 (1968).