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Detection of Platelet-Specific Protein mRNAs in Different Megakaryoblasts Using the Reverse Transcriptase Polymerase Chain Reaction a
b
b
b
Mutsumi Yasunaga , Ryukichi Ryo , Wataru Sugano & Nobuo Yamaguchi a
Department of Laboratory Medicine, Kobe University School of Medicine and Blood Transfusion Service, Kobe University Hospital Chuo-ku, Kobe, Japan b
Department of Laboratory Medicine, Kobe University School of Medicine and Blood Transfusion Service Chuo-ku, Kobe, Japan Published online: 01 Jun 2015.
To cite this article: Mutsumi Yasunaga, Ryukichi Ryo, Wataru Sugano & Nobuo Yamaguchi (1992) Detection of PlateletSpecific Protein mRNAs in Different Megakaryoblasts Using the Reverse Transcriptase Polymerase Chain Reaction, Leukemia & Lymphoma, 7:5-6, 505-510, DOI: 10.3109/10428199209049808 To link to this article: http://dx.doi.org/10.3109/10428199209049808
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
0 1992 Hanvood
Academic Publishers GmbH Printed in the United Kingdom
Leukemia and Lymphoma, Vol. I , pp. 505-5 10 Reprints available directly from the publisher Photocopying permitted by license only
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Detection of Platelet-Specific Protein mRNAs in Different Megakaryobl.asts Using the Reverse Transcriptase P01ymerase Chain Reaction MUTSUMI YASUNAGA, RYUKICHI RYO*, WATARU SUGANO and NOBUO YAMAGUCHI Department of Laboratory Medicine, Kobe University School of Medicine and Blood Transfusion Service, Kobe University Hospital*, Chuo-ku, Kobe, Japan. (Received 2 Februarjl 1992)
The short segments of cDNA encoding glycoprotein (GP)Iba, GPIIb, GPIIIa and platelet factor (PF) 4 were amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) in order to characterize various types of megakaryoblasts. Cell lines with megakaryocytic features (K562, C M K and HEL) were tested. GPIbcc, GPIIb and G P l l l a mRNAs were found to be present in K562, C M K and HEL cells, while only HEL cells expressed P F 4 or mRNA. These results suggested that megakaryoblastic cell lines could be categorized into two groups, one with the PF4 transcript and the other without it. PF4 mRNA was present in the cells obtained from one Down’s syndrome patient with transient myeloproliferative disorder and in one patient with primary myelofibrosis and megakaryoblastosis. O n the other hand, one patient with acute megakaryoblastic leukemia transformed from refractory anemia had a poor prognosis with megakaryoblastic leukemia cells which expressed no PF4 mRNA. These observations suggested that the expression of PF4 mRNA in peripheral blood megakaryoblasts may indicate the absence of a true leukemic process. KEY WORDS:
RT-PCR disorder
megakaryoblastic leukemia platelet factor 4
transient myeloproliferative
synthesis of platelet fibrinogen4. In addition it has been useful in determining the molecular abnormaliThe polymerase chain reaction (PCR) is a simple ties responsible for the inherited thrombocytomethod for amplifying specific DNA sequences. The pathiesS-’ and characterizing megakaryocytes at the development of PCR technology has made molecular molecular l e ~ e l ~ . ~ . biological study easier in all fields of medical science. In this study, PCR amplification of platelet-specific Regarding the investigation of platelets and mega- protein cDNA was used to characterize various types karyocytes, PCR has been shown to be useful in of megakaryoblasts. As judged by differences among elucidating the molecular nature of platelet alloanti- monoclonal antibodies against platelet-specific progen systems’.* and examining the multiple forms of teins, the phenotypes of megakaryoblastic leukemia glycoprotein (GP)IIb mRNA3 as well as the site of cells varied from case to case. To further characterize megakaryoblastic leukemia cells, analysis of the expression of platelet-specific protein mRNA by Address for correspondence: Mutsumi; Yasunage, M.D., Department of Laboratory Medicine, Koba University School of megakaryoblastic leukemia cells is required. Accordingly, the expression of platelet-specific protein Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
INTRODUCTION
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M. YASUNAGA et a/.
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mRNAs by cells from cell lines with megakaryocytic features and from patients with megakaryoblastosis was also investigated using the reverse transcriptase (RT)-PCR. In this study we were able to demonstrate that the various types of megakaryoblastic cells could be categorized into two main groups, one with the PF4 mRNA and the other without it.
Cells
CMK17-19, HELZ0, and K562’l were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (M. A. Bioproducts, Walkersville, M.D.). The megakaryoblasts from a Down’s syndrome patient with a transient myeloproliferative disorder (TMD) from a second patient with primary myelofibrosis and a third with megakaryoblastic leukemia transformed from refractory anemia were also used for the total RNA extraction. As judged by the increased percentage of megakaryoblasts in the MATERIALS AND METHODS peripheral blood, it seemed likely that these patients could be diagnosed as having megakaryoblastic RT-PCR leukemia. Total RNA was extracted from monoTotal RNA was extracted from megakaryoblastic nuclear cells separated from heparinized venous blood leukemia cells using the guanidinium isothiocyanate by density centrifugation (Ficoll-metrizoategradient). method”. Five pg of total RNA was reverse transcribed by M-MLV reverse transcriptase (Takara Shuzo Co., Japan). Based upon the published RESULTS sequences of GPIba”, GPIIb”, GPIIIa13 and PF414 all oligonucleotides were constructed with a Gene Platelet-specific gene expression in CMK, HEL and Assembler (Pharmacia Fine Chemicals, Piscataway, K562 cells NJ). RT-PCR amplijkation These primers are shown in Table 1. Amplification Total RNA from the cell lines was reverse transcribed, of the cDNA was performed on a DNA thermal cycler and PCR was then performed. Amplification of (RPI-8000, Riko Kogaku Co., Japan) using a cDNA GPIba, GPIIb and GPIIIa cDNA was noted for all synthesis kit (Takara Shuzo)”. As not mentioned, 30 three cell lines (Figure 1, Figure 2, and Figure 3), cycles of amplification of the cDNA were usually although GPIba cDNA appeared in K562 cells only made. PCR products were identified as the correafter 60 cyles of amplification.The PCR products were sponding cDNAs using the results of Southern confirmed as the corresponding platelet-specific blotting”. protein cDNAs by Southern blot hybridization. On the other hand, RT-PCR amplification of the PF4 mRNA was noted only in HEL cells (Figure 4). PCR products for PF4 cDNA were detected in neither Southern blot analysis K562 nor CMK cells even after 60 cycles of The cDNA probes for GPIba, GPIIb, GPIIIa and amplification. PF4 were kindly supplied by Dr. Roth” and Dr. Poncz’ ’-14. Radiolabelling and hybridization were Characterization of megakaryoblasts from a Down’s syndrome patient with a transient myeloproliferative performed as described previously16. Table 1 Oligonucleotide primers used for the RT-PCR. Oligonucleotides which flank at least two different exons were synthesized using published cDNA sequences. Gene
Sense
Antisense
Size (bp)
5'-ACCTAGA AGTGA ACTGTG AC-3' (125-144)
5’-TTTTCAGCATTGTCCTGCAG-3’ (748-767) 5’-ACGTCCAGCAGCACAATCTTJ (2294-23 13) 5’-ACATGCAGGTGTCAGTAC-3’ (1 782-1799) 5‘-GGGCTTGCAGGTCCA AGCA A-3’ (246265)
643
~~
GPlba GPllb
GPllla
PF-4
5'-GGTTTG AG AG ACTCATCTG-3' (209621 15) 5’-CAGGTCACCTTTGATTGTGA-3’ ( 13761395) 5’-TTGCTGCTCCTGCCACTTGT-3’ (55-74)
218 424 211
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PLATELET-SPECIFIC PROTEIN IN MEGAKARYOBLASTS-RT-PCR
Figure 1 RT-PCR amplification of GPlba mRNA in megakaryoblastic leukemia cells. M: marker; Lane 1: the direct PCR product with the RNA obtained from CMK cells (negative control); Lane 2, Lane 3 and Lane 4: the RT-PCR products from RNA obtained from CMK, HEL and K562 cells. respectively; Lane 5: the RT-PCR products from K562 cells with 60 cycles Of amplification. The faint band was augmented by further amplification. The 643 bp bands were the expected Products of GPlba cDNA molecules.
507
Figure 3 RT-PCR amplification of GPIIla mRNA in megakaryoblastic leukemia cells. M: marker; Lane 1: the direct PCR product with the RNA obtained from K562 cells (negative control); Lane 2, Lane 3 and Lane 4: the RT-PCR products from RNA obtained from CMK, HEL and K562 cells, respectively. The 424 bp bands were the expected products of G P l l l a cDNA molecules.
Figure 2 RT-PCR amplification of G P l l b mRNA in megakaryoblastic leukemia cells. M: marker; Lane I : the direct PCR product with the RNA obtained from HEL cells (negative control); Lane 2, Lane 3 and Lane 4: the RT-PCR products from RNA obtained from CMK, HEL and K562 cells, respectively. The 218 bp bands were the expected products of G P l l b cDNA molecules.
Figure 4 RT-PCR amplification of PF4 mRNA in megakaryoblastic leukemia cells. M: marker, Lane 1: the direct PCR from the RNA obtained from C M K cells (negative control), Lane 2. Lane 3 and Lane 4: the RT-PCR products from RNA obtained from CMK, HEL and K562 cells, respectively. The 211 bp bands were the expected products of PF4 cDNA molecules.
disorder, from a patient with primary myelofibrosis, and from a case with megakaryoblastic leukemia transformed from refractory anemia.
trisomy 21 in the patient. The blasts in the peripheral blood and bone marrow were found to have megakaryocytic characteristics, as judged by their reactivity with the monoclonal antibodies against CD4la and CD42b22. May-Giemsa staining and detection of platelet peroxidase activity confirmed that these blasts were megakaryoblasts. The patient was alive for at least 6 months following the
RT-PCR of the PF4 mRNA: Down's syndrome with TMD Laboratory data for case 1 are shown in Table 2. Chromosome analysis demonstrated the presence of
M. YASUNAGA er al.
508 Table 2 Laboratory data for the Down’s syndrome patient with TMD Case
WBC ( X 103jmmJ RBC ( x 1 0 4 / ~ ~ 3 ’ Hb(g/dl) Platelet ( x 104/mm3) Blast (Yo) CD4la (YO) CD42b (Yo) PPO
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Bone marrow NCC ( x 104/mm3) Blast (YO)
I
68.8 513 19.6 49.6 17 25.32 59.10
(+I 32.6 46
Chromozome
47XY (+21)
disappearance of megakaryoblasts in the peripheral blood. RT-PCR analysis demonstrated that PF4 mRNA was present in these megakaryoblasts (Figure 5).
Myelojibrosis The patient was a 67-year-old woman who was first diagnosed as having Banti’s syndrome and had undergone splenectomy in 1971. Severe anemia associated with an increased leukocyte and platelet count was noted in 1985. Thereafter, administration of an alkylating agent was begun in order to control the increase in the number of hemapoietic cells. A
bone marrow aspirate specimen could not be obtained (dry tap) and bone marrow biopsy showed marked myelofibrosis. In February 1989, megakaryoblasts and micromegakaryocytes appeared in the patient’s peripheral blood, suggesting evolution to megakaryoblastic leukemia. However, the patient has remained clinically well for at least 24 months following this suspected “leukemic crisis”. The clinical course of this patient with atypical primary myelofibrosis is illustrated in Figure 6. As shown in Figure 5, the PF4 mRNA was also found to be present in the mononuclear cells examined in March 1991. Refractory anemia terminating in acute megakaryoblastic leukemia The patient was a 72-year-old man who developed overt megakaryoblastic leukemia following prior refractory anemia. His clinical course has been previously described in our earlier paperz3. The patient died about 3 months after clonal evolution to megakaryoblastic leukemia. As shown in Figure 5, no PF4 mRNA was detected in a sample of mononuclear cells, obtained in September 1986, containing about 80% megakaryoblasts.
Busulfan
Mithbronitol
11.5WF1
5owaO
1
M
u
U’
III
Figure 5 RT-PCR amplification of PF4 mRNA in the cells obtained from the patients with TMD disorder, atypical primary myelofibrosis and overt megakaryoblastic leukemia transformed from refractory anemia. The 211 bp bands were the expected products of PF4 cDNA molecules. Case I : a Down’s syndrome patient with TMD; Case 2: the patient with atypical myelofibrosis; Case 3: the patient with overt megakarvoblastic leukemia transformed from refractory anemia; M: marker. I
_
3001 ~
100
WBC (X10Ymm3)
I
500
1
300 100 -
blastoid cell
Platelet (x104/mm3)
‘:;Iw
rnn
A.
\
1
I
1975
1980
1985
1990
Figure 6 Clinical course of the 62-vear-old uatient with atvuical pr&ary myelofibrosis. I .
PLATELET-SPECIFIC PROTEIN IN MEGAKARYOBLASTS-RT-PCR
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DISCUSSION It is generally accepted that megakaryoblasts can be characterized by their reactivity with monoclonal antibodies against platelet proteins and by electron microscopic ultrastructural e ~ a m i n a t i o nHowever, ~~. since platelet-specific cellular antigens should appear only after transcription of the corresponding mRNA, it can be assumed that analysis at the mRNA level may be more sensitive for characterizing megakaryoblasts. Recent studies have clearly shown that the use of RT-PCR is the most sensitive method for the detection of mRNA in cells. This technique was therefore used in this study to characterize various types of the megakaryoblasts at the molecular level. We first examined the expression of GPIba, GPIIb, GPIIIa and PF4 mRNAs in the cell lines with megakaryocytic features. Our present studies also revealed that the RT-PCR technique was useful to detect small quantities of GPIbcr and GPIIb mRNAs in K562 cells which exhibited neither GPIbcr nor GPIIb antigens2’. Our previous report has also showed that neither GPIbcc nor GPIIb mRNAs were detectable in K562 cells using Northern blot analysis ~ ~ . then found that the and in situ h y b r i d i ~ a t i o n We RT-PCR technique was the most sensitive method for the detection of mRNA encoding platelet-specific proteins in megakaryoblasts. In addition, it was found that GPIbcr, GPIIb and GPIIIa mRNAs were present in K562, CMK and HEL cells, while only HEL cells expressed PF4 mRNA. Based on the data obtained from this RT-PCR analysis, the cell lines with megakaryocytic features could be categorized into two main groups, one with the PF4 mRNA (HEL) and the other without it (K562 and CMK). The synthesis of PF4 occurs later than that of the surface GPs during megakaryocyte Moreover, the results of our previous study3’ indicated that the expression of PF4 mRNA by megakaryoblasts may be a good marker for the identification of the mature megakaryoblast. In order to test this hypothesis, expression of the PF4 mRNA in patients with an increased numbers of megakaryoblasts was determined using the RT-PCR method. The PF4 mRNA was detected in cells obtained from the patient with TMD. In this respect it is well known that a transient increase of megakaryoblasts in some neonates with Down’s syndrome can spontaneously resolve without chemotherapy. Our patient also showed the typical resolution of this increase in the number of observed megakaryoblasts. Thus, the expression of PF4 mRNA
509
in the megakaryoblasts in our patient with T M D may be evidence of the fact that the megakaryoblasts present in association with T M D are in fact benign and more mature. PF4 mRNA was also present in cells obtained from the patient with atypical myelofibrosis. Because of her unusual clinical course, it seems likely that the megakaryoblasts in this particular patient with primary myelofibrosis and suspected megakaryocytic leukemic crisis may not actually have been leukemic in nature. The presence of PF4 mRNA expression in the cells isolated from this patient seems to be consistent with our hypothesis. The detection of PF4 mRNA in these two patients with a good prognosis confirms the hypothesis we proposed previously3’ namely, that patients with an increase in the number of megakaryoblasts expressing PF4 mRNA will generally have a long survival and a good response to chemotherapy. In this respect it is noteworthy that the megakaryoblasts from a patient with overt megakaryoblastic leukemia with a poor prognosis, were found not to express any PF4 mRNA. The nature of the differences between megakaryoblasts with and without PF4 mRNA still remains to be clarified. However, the results of the present study suggest that the detection of PF4 mRNA by RT-PCR may be a useful guide in the characterization of megakaryoblasts. In addition, it seems possible that the presence of PF4 mRNA expression in megakaryoblasts, suggests that they are in fact mature and not leukemic in nature.
REFERENCES 1. Newman, P. J., Derbes, R. S. and Aster, R. H. (1989).The human
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platelet alloantigens, PIA’ and PIA2,are associated with a l e ~ c i n e ~ ~ / p r o l iamino n e ~ ~ acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing. J. Clin. Invest., 83, 1778-1781. Lynman, S., Aster, R. H., Visentin, G. P. and Newman, P. J. (1990). Polymorphism of human platelet membrane gl_yco; protein Ilb associated with the Bak”/Bakb alloantigen system. Blood, 75, 2343-2348. Bray, P. F., Leung, C. S. and Shuman, M. A. (1990). Human platelets and megakaryocytes contain alternately spliced glycoprotein IIb mRNAs. J. Biol. Chem., 265, 9587-9590. Louache, F., Debili, N., Cramer, E., Breton-Gorius, J. and Vainchenker, W. (1991). Fibrinogen is not synthesized by human megakaryocytes. Blood, 77, 31 1-316. Finch, C. N., Miller, J. L., Lyle, V. A. and Handin, R. 1. (1990). Evidence that an abnormality in the glycoprotein Ib alpha gene is not the cause of abnormal platelet function in a family with classic Bernard-Soulier disease. Blood, 75, 2357-2362. Burk, C. D., Newman, P. J., Lyman, S., Gill, J., Coller, B. S. and Poncz, M. (1991). A deletion in the gene for glycoprotein Ilb associated with Glanzmann’s thrombasthenia. J. Clin. Invest., 87, 27&276.
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7. Yasunaga, M., Ryo, R., Adachi, M., Sugano, W., Yoshida, A., Nakayama, K., Saigo, K.,Yasunaga, K. and Yamaguchi, N. (1992).Analysis of the GPIIb and GPIIla genes in the patients with Glanzmann's thrombasthenia. Jpn. J. Clin. Hematol., 33, 23&237. 8. Navarro, S., Debili, N., Couedic, J. P. L., Klein, B., Breton-Gorius, J., Doly, J. and Vainchenker, W. (1991). Interleukin-6 and its receptor are expressed by human megakaryocytes: In vitro effects on proliferation and endoreplication. Blood, 77, 461-471. 9. Adachi, M., Yasunaga, M., Nakayama, K., Jikai, J., Yamaguchi, N. and Ryo, R. (1991). Expression and detection of platelet-specific genes in human megakaryocytic cells. Jpn. J. Clin. Pathol., 39, 1308-1 316. 10. Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem., 162, 1 5 6 159. 11. Lopez, J. A,, Chung, D. W., Fujikawa, K., Hagen, F. S., Papayannoulou, T. and Roth, G. J. (1987). Cloning of the a chain of human platelet glycoprotein Ib: A transmembrane protein with homology to leucine-rich a2-glycoprotein. Proc. Narl. Acad. Sci. USA., 84, 5615-5619. 12. Heidenreich, R., Eisman, R., Surrey, S., Delgrosso, K., Bennet, J. S., Schwartz, E. and Proncz, M. (1990). Organization of the gene for platelet glycoprotein Ilb. Biochemistry, 29,1232-1244. 13. Zimrin, A. B., Gidwitz, S., Lord, S., Schwartz, E., Bennett, J. S., White, G. C. and Proncz, M. (1990). The genomic organization of platelet glycoprotein Illa. J. Biol. Chem., 265, 859&8595. 14. Eisman, R., Surrey, S., Ramachandran, B., Schwartz, E. and Poncz, M. (1990). Structural and functional comparison of the genes for human platelet factor 4 and PF4 ah. Blood, 76, 336344. 15. Yasunaga, M., Ryo, R. and Yamaguchi, N. (1991). RT-PCR amplification of platelet-specific protein mRNA. Jpn. J. Thromb. Hemosr., 2, 336340. 16. Adachi, M., Ryo, R., Yoshida, A., Sugano, W., Yasunaga, M.. Yamaguchi, N., Sato, T., Sano, K., Kaibuchi, K. and Takai, Y. (1992). Induction of the smg P2l/raplA/Krev-l p21 gene expression during the phorbol ester-induced differentiation of a human megakaryocytic leukemia cell line. Oncogene, 7, 323-329. 17. Ryo, R., Yoshida, A., Adachi, M., Sugano, W., Yasunaga, M., Yoneda, N., Yamaguchi, N. and Sato, T. (1990). Cytosolic calcium mobilization and thromboxane synthesis in a human megakaryocytic leukemia cell. ESP. Hemurol., 18, 271-275. 18. Yoshida, A., Ryo, R., Yamaguchi, N. and Okuma, M. (1992). Expression of P62, a putative collagen receptor, on human megakaryocytic leukemia cells. Leukemia and Lymphoma, 6, I9 1-196. 19. Adachi, M., Ryo, R., Yoshida, A., Teshigawara, K., Yamaguchi,
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N., Hoshijima, M., Takai, Y. and Sato, T. (1989). Elevation of intracellular calcium ion by prostaglandin El and its inhibition by protein kinase C in a human megakaryocyte leukemia cell line. Cancer Res., 49, 3805-3808. Uzan, G., Frachet, P., Lajmanovich, A,, Prandini, M. H., Denarier, E., Duperray, A,, Loftus, J., Ginsberg, M., Plow, E. and Marguerie, G. (1988). cDNA clones for human platelet GPllb corresponding to mRNA from megakaryocytes and HEL cells. Eur. J. Biochem., 171, 87-93. Adachi, M., Ryo, R., Yoshida, A,, Yoneda, N., Tatsumi, E., Yamaguchi, N. and Kishimoto, Y. (1989). Kinetic study of platelet glycoproteins and PPO activity in TPA-induced K562 cells. Acta Haemat. Jpn., 51, 102-1 12. Yoshioka, M., Ryo, R., Hirai, K., Yasunaga, K. and Yamaguchi, N. (1988). Preparation of platelet G P Ilb/lIla complex monoclonal antibody and its clinical application. Acru. Haemat. Jpn., 51, 1138-1146. Adachi, M., Ryo, R., Yoshida, A,, Yamaguchi, N. and Izumi, Y. (1989). Refractory anemia terminating in acute megakaryoblastic leukemia (M7). Acra. Haemat. (Basel). 81, I04108. Vainchenker, W., Deschamps, J. F., Bastin, J. M., Guichard, M., Titeux, M., Breton-Gorius, J. and McMichael, J. (1982). Two monoclonal anti-platelet antibodies as markers of human megakaryocyte maturation: Immunofluorescence staining and platelet peroxidase detection in megakaryocyte colonies and in vivo cells from normal and leukemic patients. Blood, 59, 514521. Jikai, J., Ryo, R., Yasunaga, M., Sugano, W., Adachi, M., Yamaguchi, N. and Hirano, T. Detection of platelet-specific mRNA using in situ hybridization. Jpn. J. Clin. Parhol., in press. Ryo, R., Proffitt, R. T., Poger, M. E., O'Bear, R. and Deuel, T. F. (1980). Human platelet factor 4 antigen in megakaryocytes. Thromb. Res., 17, 645-652. Ryo, R., Nakeff, A,, Huang, S. S., Ginsberg, M. and Deuel, T. F. (1983). New synthesis of platelet-specific protein: Platelet factor 4 synthesis in a megakaryocyte-enriched rabbit bone marrow culture system. J. Cell. Biol., %, 515-520. Ryo, R., Yoshida, A. and Yamaguchi, N. (1990). Megakaryocytic leukemia cell lines and megakaryocytic leukemia. Jpn. J. Clin. Pathol., 38, 5 14-523. Adachi, M., Ryo, R., Sato, T. and Yamaguchi, N. (1991).Platelet factor 4 gene expression in a human megakaryocytic leukemia cell line (CMK) and its differentiated subclone(CMK 11-5).ESP. Hematol., 19, 923-927. Ryo, R., Adachi, M., Sugano, W., Yasunaga, M., Yoshida, A,, Jikai, J., Yamaguchi, N., Akita, H., Yokoyama, M.. Konaka, T. and Poncz, M. (1991). Platelet factor 4 mRNA expression in cells from a patient with megakaryoblastic crisis in chronic myelogeneous leukemia. Cancer, 67,96&964.
Leukemia & Lymphoma Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ilal20
Detection of Platelet-Specific Protein mRNAs in Different Megakaryoblasts Using the Reverse Transcriptase Polymerase Chain Reaction a
b
b
b
Mutsumi Yasunaga , Ryukichi Ryo , Wataru Sugano & Nobuo Yamaguchi a
Department of Laboratory Medicine, Kobe University School of Medicine and Blood Transfusion Service, Kobe University Hospital Chuo-ku, Kobe, Japan b
Department of Laboratory Medicine, Kobe University School of Medicine and Blood Transfusion Service Chuo-ku, Kobe, Japan Published online: 01 Jun 2015.
To cite this article: Mutsumi Yasunaga, Ryukichi Ryo, Wataru Sugano & Nobuo Yamaguchi (1992) Detection of PlateletSpecific Protein mRNAs in Different Megakaryoblasts Using the Reverse Transcriptase Polymerase Chain Reaction, Leukemia & Lymphoma, 7:5-6, 505-510, DOI: 10.3109/10428199209049808 To link to this article: http://dx.doi.org/10.3109/10428199209049808
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
0 1992 Hanvood
Academic Publishers GmbH Printed in the United Kingdom
Leukemia and Lymphoma, Vol. I , pp. 505-5 10 Reprints available directly from the publisher Photocopying permitted by license only
Downloaded by [University of Saskatchewan Library] at 13:17 13 August 2015
Detection of Platelet-Specific Protein mRNAs in Different Megakaryobl.asts Using the Reverse Transcriptase P01ymerase Chain Reaction MUTSUMI YASUNAGA, RYUKICHI RYO*, WATARU SUGANO and NOBUO YAMAGUCHI Department of Laboratory Medicine, Kobe University School of Medicine and Blood Transfusion Service, Kobe University Hospital*, Chuo-ku, Kobe, Japan. (Received 2 Februarjl 1992)
The short segments of cDNA encoding glycoprotein (GP)Iba, GPIIb, GPIIIa and platelet factor (PF) 4 were amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) in order to characterize various types of megakaryoblasts. Cell lines with megakaryocytic features (K562, C M K and HEL) were tested. GPIbcc, GPIIb and G P l l l a mRNAs were found to be present in K562, C M K and HEL cells, while only HEL cells expressed P F 4 or mRNA. These results suggested that megakaryoblastic cell lines could be categorized into two groups, one with the PF4 transcript and the other without it. PF4 mRNA was present in the cells obtained from one Down’s syndrome patient with transient myeloproliferative disorder and in one patient with primary myelofibrosis and megakaryoblastosis. O n the other hand, one patient with acute megakaryoblastic leukemia transformed from refractory anemia had a poor prognosis with megakaryoblastic leukemia cells which expressed no PF4 mRNA. These observations suggested that the expression of PF4 mRNA in peripheral blood megakaryoblasts may indicate the absence of a true leukemic process. KEY WORDS:
RT-PCR disorder
megakaryoblastic leukemia platelet factor 4
transient myeloproliferative
synthesis of platelet fibrinogen4. In addition it has been useful in determining the molecular abnormaliThe polymerase chain reaction (PCR) is a simple ties responsible for the inherited thrombocytomethod for amplifying specific DNA sequences. The pathiesS-’ and characterizing megakaryocytes at the development of PCR technology has made molecular molecular l e ~ e l ~ . ~ . biological study easier in all fields of medical science. In this study, PCR amplification of platelet-specific Regarding the investigation of platelets and mega- protein cDNA was used to characterize various types karyocytes, PCR has been shown to be useful in of megakaryoblasts. As judged by differences among elucidating the molecular nature of platelet alloanti- monoclonal antibodies against platelet-specific progen systems’.* and examining the multiple forms of teins, the phenotypes of megakaryoblastic leukemia glycoprotein (GP)IIb mRNA3 as well as the site of cells varied from case to case. To further characterize megakaryoblastic leukemia cells, analysis of the expression of platelet-specific protein mRNA by Address for correspondence: Mutsumi; Yasunage, M.D., Department of Laboratory Medicine, Koba University School of megakaryoblastic leukemia cells is required. Accordingly, the expression of platelet-specific protein Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
INTRODUCTION
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M. YASUNAGA et a/.
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mRNAs by cells from cell lines with megakaryocytic features and from patients with megakaryoblastosis was also investigated using the reverse transcriptase (RT)-PCR. In this study we were able to demonstrate that the various types of megakaryoblastic cells could be categorized into two main groups, one with the PF4 mRNA and the other without it.
Cells
CMK17-19, HELZ0, and K562’l were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (M. A. Bioproducts, Walkersville, M.D.). The megakaryoblasts from a Down’s syndrome patient with a transient myeloproliferative disorder (TMD) from a second patient with primary myelofibrosis and a third with megakaryoblastic leukemia transformed from refractory anemia were also used for the total RNA extraction. As judged by the increased percentage of megakaryoblasts in the MATERIALS AND METHODS peripheral blood, it seemed likely that these patients could be diagnosed as having megakaryoblastic RT-PCR leukemia. Total RNA was extracted from monoTotal RNA was extracted from megakaryoblastic nuclear cells separated from heparinized venous blood leukemia cells using the guanidinium isothiocyanate by density centrifugation (Ficoll-metrizoategradient). method”. Five pg of total RNA was reverse transcribed by M-MLV reverse transcriptase (Takara Shuzo Co., Japan). Based upon the published RESULTS sequences of GPIba”, GPIIb”, GPIIIa13 and PF414 all oligonucleotides were constructed with a Gene Platelet-specific gene expression in CMK, HEL and Assembler (Pharmacia Fine Chemicals, Piscataway, K562 cells NJ). RT-PCR amplijkation These primers are shown in Table 1. Amplification Total RNA from the cell lines was reverse transcribed, of the cDNA was performed on a DNA thermal cycler and PCR was then performed. Amplification of (RPI-8000, Riko Kogaku Co., Japan) using a cDNA GPIba, GPIIb and GPIIIa cDNA was noted for all synthesis kit (Takara Shuzo)”. As not mentioned, 30 three cell lines (Figure 1, Figure 2, and Figure 3), cycles of amplification of the cDNA were usually although GPIba cDNA appeared in K562 cells only made. PCR products were identified as the correafter 60 cyles of amplification.The PCR products were sponding cDNAs using the results of Southern confirmed as the corresponding platelet-specific blotting”. protein cDNAs by Southern blot hybridization. On the other hand, RT-PCR amplification of the PF4 mRNA was noted only in HEL cells (Figure 4). PCR products for PF4 cDNA were detected in neither Southern blot analysis K562 nor CMK cells even after 60 cycles of The cDNA probes for GPIba, GPIIb, GPIIIa and amplification. PF4 were kindly supplied by Dr. Roth” and Dr. Poncz’ ’-14. Radiolabelling and hybridization were Characterization of megakaryoblasts from a Down’s syndrome patient with a transient myeloproliferative performed as described previously16. Table 1 Oligonucleotide primers used for the RT-PCR. Oligonucleotides which flank at least two different exons were synthesized using published cDNA sequences. Gene
Sense
Antisense
Size (bp)
5'-ACCTAGA AGTGA ACTGTG AC-3' (125-144)
5’-TTTTCAGCATTGTCCTGCAG-3’ (748-767) 5’-ACGTCCAGCAGCACAATCTTJ (2294-23 13) 5’-ACATGCAGGTGTCAGTAC-3’ (1 782-1799) 5‘-GGGCTTGCAGGTCCA AGCA A-3’ (246265)
643
~~
GPlba GPllb
GPllla
PF-4
5'-GGTTTG AG AG ACTCATCTG-3' (209621 15) 5’-CAGGTCACCTTTGATTGTGA-3’ ( 13761395) 5’-TTGCTGCTCCTGCCACTTGT-3’ (55-74)
218 424 211
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PLATELET-SPECIFIC PROTEIN IN MEGAKARYOBLASTS-RT-PCR
Figure 1 RT-PCR amplification of GPlba mRNA in megakaryoblastic leukemia cells. M: marker; Lane 1: the direct PCR product with the RNA obtained from CMK cells (negative control); Lane 2, Lane 3 and Lane 4: the RT-PCR products from RNA obtained from CMK, HEL and K562 cells. respectively; Lane 5: the RT-PCR products from K562 cells with 60 cycles Of amplification. The faint band was augmented by further amplification. The 643 bp bands were the expected Products of GPlba cDNA molecules.
507
Figure 3 RT-PCR amplification of GPIIla mRNA in megakaryoblastic leukemia cells. M: marker; Lane 1: the direct PCR product with the RNA obtained from K562 cells (negative control); Lane 2, Lane 3 and Lane 4: the RT-PCR products from RNA obtained from CMK, HEL and K562 cells, respectively. The 424 bp bands were the expected products of G P l l l a cDNA molecules.
Figure 2 RT-PCR amplification of G P l l b mRNA in megakaryoblastic leukemia cells. M: marker; Lane I : the direct PCR product with the RNA obtained from HEL cells (negative control); Lane 2, Lane 3 and Lane 4: the RT-PCR products from RNA obtained from CMK, HEL and K562 cells, respectively. The 218 bp bands were the expected products of G P l l b cDNA molecules.
Figure 4 RT-PCR amplification of PF4 mRNA in megakaryoblastic leukemia cells. M: marker, Lane 1: the direct PCR from the RNA obtained from C M K cells (negative control), Lane 2. Lane 3 and Lane 4: the RT-PCR products from RNA obtained from CMK, HEL and K562 cells, respectively. The 211 bp bands were the expected products of PF4 cDNA molecules.
disorder, from a patient with primary myelofibrosis, and from a case with megakaryoblastic leukemia transformed from refractory anemia.
trisomy 21 in the patient. The blasts in the peripheral blood and bone marrow were found to have megakaryocytic characteristics, as judged by their reactivity with the monoclonal antibodies against CD4la and CD42b22. May-Giemsa staining and detection of platelet peroxidase activity confirmed that these blasts were megakaryoblasts. The patient was alive for at least 6 months following the
RT-PCR of the PF4 mRNA: Down's syndrome with TMD Laboratory data for case 1 are shown in Table 2. Chromosome analysis demonstrated the presence of
M. YASUNAGA er al.
508 Table 2 Laboratory data for the Down’s syndrome patient with TMD Case
WBC ( X 103jmmJ RBC ( x 1 0 4 / ~ ~ 3 ’ Hb(g/dl) Platelet ( x 104/mm3) Blast (Yo) CD4la (YO) CD42b (Yo) PPO
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Bone marrow NCC ( x 104/mm3) Blast (YO)
I
68.8 513 19.6 49.6 17 25.32 59.10
(+I 32.6 46
Chromozome
47XY (+21)
disappearance of megakaryoblasts in the peripheral blood. RT-PCR analysis demonstrated that PF4 mRNA was present in these megakaryoblasts (Figure 5).
Myelojibrosis The patient was a 67-year-old woman who was first diagnosed as having Banti’s syndrome and had undergone splenectomy in 1971. Severe anemia associated with an increased leukocyte and platelet count was noted in 1985. Thereafter, administration of an alkylating agent was begun in order to control the increase in the number of hemapoietic cells. A
bone marrow aspirate specimen could not be obtained (dry tap) and bone marrow biopsy showed marked myelofibrosis. In February 1989, megakaryoblasts and micromegakaryocytes appeared in the patient’s peripheral blood, suggesting evolution to megakaryoblastic leukemia. However, the patient has remained clinically well for at least 24 months following this suspected “leukemic crisis”. The clinical course of this patient with atypical primary myelofibrosis is illustrated in Figure 6. As shown in Figure 5, the PF4 mRNA was also found to be present in the mononuclear cells examined in March 1991. Refractory anemia terminating in acute megakaryoblastic leukemia The patient was a 72-year-old man who developed overt megakaryoblastic leukemia following prior refractory anemia. His clinical course has been previously described in our earlier paperz3. The patient died about 3 months after clonal evolution to megakaryoblastic leukemia. As shown in Figure 5, no PF4 mRNA was detected in a sample of mononuclear cells, obtained in September 1986, containing about 80% megakaryoblasts.
Busulfan
Mithbronitol
11.5WF1
5owaO
1
M
u
U’
III
Figure 5 RT-PCR amplification of PF4 mRNA in the cells obtained from the patients with TMD disorder, atypical primary myelofibrosis and overt megakaryoblastic leukemia transformed from refractory anemia. The 211 bp bands were the expected products of PF4 cDNA molecules. Case I : a Down’s syndrome patient with TMD; Case 2: the patient with atypical myelofibrosis; Case 3: the patient with overt megakarvoblastic leukemia transformed from refractory anemia; M: marker. I
_
3001 ~
100
WBC (X10Ymm3)
I
500
1
300 100 -
blastoid cell
Platelet (x104/mm3)
‘:;Iw
rnn
A.
\
1
I
1975
1980
1985
1990
Figure 6 Clinical course of the 62-vear-old uatient with atvuical pr&ary myelofibrosis. I .
PLATELET-SPECIFIC PROTEIN IN MEGAKARYOBLASTS-RT-PCR
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DISCUSSION It is generally accepted that megakaryoblasts can be characterized by their reactivity with monoclonal antibodies against platelet proteins and by electron microscopic ultrastructural e ~ a m i n a t i o nHowever, ~~. since platelet-specific cellular antigens should appear only after transcription of the corresponding mRNA, it can be assumed that analysis at the mRNA level may be more sensitive for characterizing megakaryoblasts. Recent studies have clearly shown that the use of RT-PCR is the most sensitive method for the detection of mRNA in cells. This technique was therefore used in this study to characterize various types of the megakaryoblasts at the molecular level. We first examined the expression of GPIba, GPIIb, GPIIIa and PF4 mRNAs in the cell lines with megakaryocytic features. Our present studies also revealed that the RT-PCR technique was useful to detect small quantities of GPIbcr and GPIIb mRNAs in K562 cells which exhibited neither GPIbcr nor GPIIb antigens2’. Our previous report has also showed that neither GPIbcc nor GPIIb mRNAs were detectable in K562 cells using Northern blot analysis ~ ~ . then found that the and in situ h y b r i d i ~ a t i o n We RT-PCR technique was the most sensitive method for the detection of mRNA encoding platelet-specific proteins in megakaryoblasts. In addition, it was found that GPIbcr, GPIIb and GPIIIa mRNAs were present in K562, CMK and HEL cells, while only HEL cells expressed PF4 mRNA. Based on the data obtained from this RT-PCR analysis, the cell lines with megakaryocytic features could be categorized into two main groups, one with the PF4 mRNA (HEL) and the other without it (K562 and CMK). The synthesis of PF4 occurs later than that of the surface GPs during megakaryocyte Moreover, the results of our previous study3’ indicated that the expression of PF4 mRNA by megakaryoblasts may be a good marker for the identification of the mature megakaryoblast. In order to test this hypothesis, expression of the PF4 mRNA in patients with an increased numbers of megakaryoblasts was determined using the RT-PCR method. The PF4 mRNA was detected in cells obtained from the patient with TMD. In this respect it is well known that a transient increase of megakaryoblasts in some neonates with Down’s syndrome can spontaneously resolve without chemotherapy. Our patient also showed the typical resolution of this increase in the number of observed megakaryoblasts. Thus, the expression of PF4 mRNA
509
in the megakaryoblasts in our patient with T M D may be evidence of the fact that the megakaryoblasts present in association with T M D are in fact benign and more mature. PF4 mRNA was also present in cells obtained from the patient with atypical myelofibrosis. Because of her unusual clinical course, it seems likely that the megakaryoblasts in this particular patient with primary myelofibrosis and suspected megakaryocytic leukemic crisis may not actually have been leukemic in nature. The presence of PF4 mRNA expression in the cells isolated from this patient seems to be consistent with our hypothesis. The detection of PF4 mRNA in these two patients with a good prognosis confirms the hypothesis we proposed previously3’ namely, that patients with an increase in the number of megakaryoblasts expressing PF4 mRNA will generally have a long survival and a good response to chemotherapy. In this respect it is noteworthy that the megakaryoblasts from a patient with overt megakaryoblastic leukemia with a poor prognosis, were found not to express any PF4 mRNA. The nature of the differences between megakaryoblasts with and without PF4 mRNA still remains to be clarified. However, the results of the present study suggest that the detection of PF4 mRNA by RT-PCR may be a useful guide in the characterization of megakaryoblasts. In addition, it seems possible that the presence of PF4 mRNA expression in megakaryoblasts, suggests that they are in fact mature and not leukemic in nature.
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