The present study was undertaken to provide information on the nucleolar and cytoplasmic density in specimens stained for RNA during "cell dedifferentiation" represented by blastic transformation of mature T lymphocytes. Nucleolar and cytoplasmic RNA's were visualized using a simple cytochemical method followed by computer assisted densitometry and size measurements of digitised images. An increased nucleolar and cytoplasmic RNA density accompanying the blastic transformation was significant after 48 hours of cultivation with phytohemaglutinin (PHA) when stimulated cells were characterized the largest nucleolar size reflecting S or G2 phase of the cell cycle. On the other hand, significantly larger ratio of the nucleolar to cytoplasmic density was noted only after a shorter cultivation when stimulated cells were presumably in the G1 phase. Thus the increased nucleolar and cytoplasmic RNA density together represented an accompanying phenomenon of the cell proliferation and cycling state. From the methodical point of view, the nucleolar and cytoplasmic RNA densitometry appeared as a simple as well as useful additional method to study "dedifferentiation" or various cell states at the single cell level. In addition, it was also interesting that the increase of the nucleolar diameter in stimulated cells was much larger than that of the nucleolar density. Such difference suggested that the RNA content in nucleoli was related mainly to their size

Ilona JirĂĄskovĂĄ

Ivan Kalousek

Karel Smetana

Petra OtevrelovĂĄ

Folia Histochemica et Cytobiologica

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©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2008:46(4): 429 (429-432) doi: 10.2478/v10042-008-0074-8IntroductionIt is generally known that human mature resting Tlymphocytes stimulated with phytohemaglutinin(PHA) "dedifferentiate" and transform back to prolif-erating blastic stage [1,3]. Such transformation ofsmall mature resting to large cycling blastic cells isaccompanied by nucleolar enlargement, which is relat-ed to the increased nucleolar biosynthetic activities ofstimulated cells [1,3-5,24]. In addition, ring shapednucleoli transform to large nucleoli with a relativelyuniform distribution of RNA [1,15,18]. On the otherhand, the information on the nucleolar and cytoplas-mic RNA density in the course of blastic transforma-tion is missing although it seems to be likely that itmight be different.  The present study was undertaken to provide moreinformation on the nucleolar and cytoplasmic densityin specimens stained for RNA during "cell dedifferen-tiation" represented by blastic transformation ofmature T lymphocytes. Nucleolar and cytoplasmicRNA were visualized using a simple cytochemicalmethod followed by computer assisted densitometryand nucleolar diameter measurements of digitisedimages. The results indicated that an increasing densi-ty accompanying the blastic transformation was signif-icant after 48 hours of cultivation with PHA when thecells were in S/G2 phase of the cell cycle. On the otherhand, significantly larger ratio of the nucleolar to cyto-plasmic RNA density was noted only after a shortercultivation when stimulated cells were presumably inthe late G1 phase.  Materials and methodsHuman lymphocytes were isolated from the peripheral blood ofhealthy volunteers using a discontinuous density gradient [2] ofFOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 46, No. 4, 2008pp. 429-432On the nucleolar and cytoplasmic RNA density during"cell dedifferentiation" represented by blastic transformation of human mature T lymphocytes – a cytochemical studyKarel Smetana, Petra Otevøelová, Ilona Jirásková, Ivan Kalousek Institute of Hematology and Blood Transfusion, Prague, Czech RepublicAbstract: The present study was undertaken to provide information on the nucleolar and cytoplasmic density in specimensstained for RNA during "cell dedifferentiation" represented by blastic transformation of mature T lymphocytes. Nucleolarand cytoplasmic RNA´s were visualized using a simple cytochemical method followed by computer assisted densitometryand size measurements of digitised images. An increased nucleolar and cytoplasmic RNA density accompanying the blas-tic transformation was significant after 48 hours of cultivation with phytohemaglutinin (PHA) when stimulated cells werecharacterized the largest nucleolar size reflecting S or G2 phase of the cell cycle. On the other hand, significantly larger ratioof the nucleolar to cytoplasmic density was noted only after a shorter cultivation when stimulated cells were presumably inthe G1 phase. Thus the increased nucleolar and cytoplasmic RNA density together represented an accompanying phenom-enon of the cell proliferation and cycling state. From the methodical point of view, the nucleolar and cytoplasmic RNA den-sitometry appeared as a simple as well as useful additional method to study "dedifferentiation" or various cell states at thesingle cell level. In addition, it was also interesting that the increase of the nucleolar diameter in stimulated cells was muchlarger than that of the nucleolar density. Such difference suggested that the RNA content in nucleoli was related mainly totheir size.  Key words: nucleolar and cytoplasmic density, lymphocytes, blastic transformation  Correspondence: K. Smetana, Institute of Hematology andBlood Transfusion, U nemocnice 1, Prague 2, Czech Republic128 20; fax.: (+4202) 71977249, e-mail: karel.smetana@uhkt.czHistopaque (Sigma, St. Louis, MO USA). Cells, 1×106/ml, weresuspended in medium RPMI 1640 supplemented with 10% heat-inactivated FBS and stimulated with 10 μg/ml PHA-P from Sigmaat 37°C in a 5% CO2 humidified atmosphere for time frames up to48 hrs [8,12]. Then, harvested cells were prepared for the lightmicroscopic observations using a Shandon Cytospin 2 cytocen-trifuge (Shandon Southern Products, UK) – 800 rpm for 10 min.and unfixed cytospins were stained for RNA with acidified meth-ylene blue at pH=5.3 [13,21]. Micrographs were captured with a Camedia digital photo cam-era C.4040 ZOOM (Olympus, Japan) placed on Jenalumar micro-scope (Zeiss, Germany) equipped with the double adapter to pro-vide a larger magnification of resulting images. Then the imageswere processed and nucleolar size was measured with Quick Pho-toprogram (Olympus, Japan). Mean nucleolar diameters weredetermined on the screen at magnification ×4300 and were basedon two measurements of the long and minor axis for each nucleo-lus [10,14,17]. It should be mentioned that the nucleolar size anddistribution of RNA facilitated to estimate the cell cycle phase ofstudied cells [6,9,11,20,23,24].  The nucleolar and cytoplasmic density in specimens stained forRNA was measured after image conversion to grey scale using theNIH Image Program – Scion for Windows (Scion Corp., USA).The density was expressed in relative arbitrary units, which werecalculated by subtracting measured density of nucleoli or cyto-plasm from the density surrounding the measured cell. Such calcu-lation and standardisation of arbitrary density units facilitated thecomparison of results in various portions of cytospins, which occa-sionally exhibited various artificial densities due to both prepara-tion and staining techniques. This approach decreased artificialmeasurements and thus provided better results than the backgroundadjusted to zero, which depended on the investigator. In contrast tothe nucleolar size measurements, the nucleolar and cytoplasmicdensities were measured at lower magnification using dry 50xobjective to decrease the resolving power of the microscope. Suchapproach facilitated to cover a larger and more uniform densitymeasurements of nucleolar and cytoplasmic regions stained forRNA. ResultsNucleolar size and types Quantitative data are in the Table 1. The nucleolar sizeduring blastic transformation gradually increased.Maximal values of mean nucleolar diameter in stimu-lated lymphocytes were noted after 48 hrs of cultiva-tion and apparently reflected S and G2 phase [6,9,11,16,20,23,24]. In resting mature lymphocytes repre-senting cells in G0 or /early G1 phase, nucleoli weresmall and mostly ring shaped (Fig. 1) [see also 3,6,18,23,24], After 24 hours of cultivation in the presence ofPHA, stimulated cells were characterised by enlargednucleoli with more or less distinct nucleolonemaswhich were described in late G1 and especially S orG2 phase of the cell cycle (Fig. 2) [see also 3,6,15,23,24]. Nucleolar and cytoplasmic density in specimensstained for RNAFor quantitative data see the Table 1. In control restinglymphocytes the nucleolar and cytoplasmic densitywas almost the same (Fig. 3). The nucleolar to cyto-plasmic density ratio was about one. In transforminglymphocytes to blastic state (Figs. 4,5), the nucleolardensity increased but such increase was significantonly after 48 hours of cultivation with PHA. On theother hand, the cytoplasmic density was apparentlysmaller at first but then significantly increased. There-fore, the increased nucleolar to cytoplasmic densityration in stimulated cells significantly increased onlyafter 24 hours of cultivation in the presence of PHA. Itwas also interesting that the nucleolar density in stim-ulated cells increased less dramatically than the nucle-olar diameter (Fig. 6). 430 K. Smetana et al.©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2008:46(4): 430 (429-432) doi: 10.2478/v10042-008-0074-8Table 1. Nucleolar and cytoplasmic density in specimens stained for RNA.Legend: # – based on more than 40 measurements for each group; ## – see ref. [22]; *– statistically different from 0 hrs using t-test (p<0.009);r – statis-tically different from 24 hours using t- test (p<0.001); § – statistically different from nucleoli using t-test (p<0.001); a – mean and standard deviation; No/Cy– ratio of nucleolar to cytoplasmic RNA density; NoD – mean nucleolar diameter. Fig. 1. Resting lymphocyte with a ring shaped nucleolus (arrowand insert), (magnification approx. ×3500). Fig. 2. Stimulatedtransformed and "dedifferentiated" blastic cell with a large nucle-olus with a more uniform distribution of RNA (arrow), whichshows nucleolonemata at larger magnification (insert), (magnifica-tion approx. ×2100).DiscussionThe relatively increased nucleolar RNA density duringblastic transformation (present study) is not surprisingand represents a further phenomenon, which is relatedto the generally known increased nucleolar RNA tran-scription [3-5,7,15,16,23]. Moreover, the known trans-formation of ring shaped nucleoli in resting lympho-cytes to enlarged nucleoli with less or more distinctnucleolonemas in transformed blastic cells is also inharmony with the increased nucleolar biosyntheticactivity and return to the cell cycle [3,4,6,7,15,20,23,24]. It should be added that the increase of the nucleo-lar diameter in stimulated cells was much larger thanthat of the nucleolar density. Such difference suggeststhat the RNA content in nucleoli depends mainly ontheir size. A similar RNA density of small and largenucleoli was also noted previously in leukaemia gran-ulocytic progenitors [19]. The delayed increase of the RNA cytoplasmic den-sity in stimulated cells seems to be interesting andresulted in an increased nucleolar to cytoplasmic RNAdensity ratio. Such delay might be easily explained bythe preceding increased nucleolar RNA transcriptionand assembly of ribosomes before their transport tocytoplasm [3,5,7,23]. On the other hand, when biosyn-thetic activities of nucleoli in resting cells arerepressed [4,6,7,15,24], the nucleolar and cytoplasmicRNA densities are similar but smaller (present results).At this occasion it should be mentioned that in stimu-lated and cycling blastic cells the nucleolar and cyto-plasmic RNA densities were also similar. However,they were significantly higher than in resting lympho-cytes. In that case, both the nucleolar and cytoplasmicbiosynthetic activities are known to substantiallyincrease in comparison with resting cells [3-5,7,9,15].From the methodical point of view the presentstudy, regardless of the interpretation, clearly demon-strated that the cytochemical RNA densitometry wasan useful procedure that might be complementary to431Nucleolar and cytoplasmic RNA density during human T lymphocyte dedifferentiation©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2008:46(4): 431 (429-432) doi: 10.2478/v10042-008-0074-8Fig. 3. Nucleolar (No – thin black line) and cytoplasmic (Cy – thick black line) RNA density in a small resting lymphocyte. Nucleolus –arrow. The lower part of the Figure contains nucleolar (No) and cytoplasmic (Cy) densitograms. Numbers in densitograms indicate den-sity values calculated by subtracting measured values from the background surrounding the investigated cell. The density scale is belowdensitogram (magnification approx. ×2200). Fig. 4. Nucleolar and cytoplasmic RNA density in a stimulated and transformed blastic cellafter 24 hours of cultivation in the presence of PHA. For other legend see Fig. 3 (magnification approx. ×1700). Fig. 5. Nucleolar andcytoplasmic RNA density in a stimulated and transformed blastic cell after 48 hours of cultivation in the presence of PHA. For other leg-end see Fig. 3 (magnification approx. ×1300). Fig. 6. Nucleolar diameter (NoD) and density (NoDn) in stimulat-ed lymphocytes. Mean values of the nucleolar diameter are multi-plied by 10 and mean values of the nucleolar density are dividedby 10. Standard deviations are in the Table 1. Note that theincrease of the nucleolar diameter was larger than that of the nucle-olar density.other methodical approaches facilitating to distinguishresting and stimulated – "dedifferentiated" cyclingcells at the single cell level. In addition, such approachappeared to be useful in smear or cytospins specimens,in which the number of investigated cells might bevery limited.  Acknowledgement: The present study was supported in part bythe Research Development Project VZ 0002373601 of the Ministryof Health of the Czech Republic.  References[ 1] Astaldi G, Lisiewicz J. Lymphocytes. Naples: Idelson; 1971.[ 2] Böyum A. Separation of leukocytes from blood and bonemarrow. Scan J Clin Lab Invest. Suppl. 1968;21:9-109. [ 3] Busch H, Smetana K. The nucleolus. New York: AcademicPress; 1970.[ 4] Carson DA. Biochemistry and function of lymphocytes andplasma cells. In: Williams WJ, Beutler E, Erslev AJ, Licht-man MA, eds.Hematology. New York: McGraw-Hill; 1983:903-908.[ 5] Cline MJ. The white cell. Cambridge: Harvard UniversityPress; 1975.[ 6] Gani R. The nucleoli of cultured human lymphocytes. ExpCell Res. 1976;97:249-258.[7 ] Henry PH, Levin MJ, Levin AS. Composition and biochem-istry of lymphocytes and plasma cells. In: Williams WJ, Beut-ler E, Erslev AJ, Rundles RW, eds. Hematology. New York:McGraw-Hill; 1972:782-790.[ 8] Kalousek I, Krizkova P. Lymphocyte mitogenic transforma-tion is accompanied by phosphorylation of the nucleolar tran-scription factor UBF. Cell Mol Biol. (Noisy-le-grand) 2000;46:163-1171.[ 9] Kwiatkowska M, Maszewski J. Functional and structural het-erogeneity of nucleoli: the dependence of the activity of trans-port of newly synthesized rRNA on nucleolar size and phase ofthe cell cycle. Folia Histochem Cytochem. 1985;23:117-126.[10] Monge JM, Val-Bernal JF, Buelta L, Garcia-Castrillo L, Asen-sio L. Selective nuclear morphometry as prognostic factor ofsurvival of renal carcinoma. Histol Histopathol. 1999;14:119-123.[11] Nagl W. Zellkern und Zellzyklen. Stuttgart: Verl. EugenUlmer; 1976[12] Nowell PC Differentiation of human leukaemic leukocytes intissue culture. Exp Cell Res. 1960;19:267-277.[13] Ochs RL. Methods used to study structure and function of thenucleolus. Methods Cell Biol. 1998;53:303-301.[14] Politi EN, Lazaris AC, Kavantzas A, Kountselini H. Compar-ison between morphometry and immunostaining of malignantcells in no-small cell lung cancer. Anal Quant Cytol Histol.2003;25:169-176.[15] Raška I, Koberna K, Malínský J, Fidlerová H, Mašata M. Thenucleolus and transcription of ribosomal genes. Biol Cell.2004;96:579-594.[16] Schnedl W, Schnedl M. Nukleolus Zahl und Grösse währenddes Zellzyklus. Z. Zellforsch. 1972;126:374-382.[17] Setala L, Lipponen P, Kosma VM, Marin S, Eskelinen M,Syrjanen K, Alhava E. Nuclear morphometry as predictor ofdisease outcome in gastric cancer. J Pathol. 1977;181:46-50. [18] Smetana K. Nucleoli in maturing blood cells. Top Rev Hae-matol. 1980;1:115-137.[19] Smetana K, Klamová H, Mikulenková D, Pluskaloví M,Hrkal Z. The nucleolar size and density in human early gran-ulocytic progenitors – myeloblasts. Europ J Histochem. 2006;50:119-124.[20] Smetana K, Kuzelova K, Zapotocky M, Starkova J, Hrkal Z,Trka J. Mean diameter of nucleolar bodies in cultured humanleukemic myeloblasts is related to the S and G2 phase of thecell cycle. Europ. J Histochem. 2007;51:269-274.[21] Smetana K, Lejnar J, Potmìšil M. A further contribution tothe demonstration of RNA and nucleoli of blood cells insmear preparations. Folia Haematol. 1969;91:381-384.[22] Smetana K, Otevøelová P, Kalousek I. Heterochromatin den-sity in the course of cell "dedifferentiation" represented byblastic transformation of human mature T lymphocytes. ApplBiomed. 2007;5:189-193. [23] Wachtler F, Schwarzacher HG. Ellinger A. The influence ofthe cell cycle ion structure and number of nucleoli in cultureshuman lymphocytes. Cell Tissue Res. 1982;225:155-163.[24] Wachtler F, Stahl HG. The nucleolus. Micron. 1983;24:473-505.Submitted: 29 November, 2007Accepted after reviews: 2 June, 2008432 K. Smetana et al.©Polish Histochemical et Cytochemical SocietyFolia Histochem Cytobiol. 2008:46(4): 432 (429-432) doi: 10.2478/v10042-008-0074-8

On the nucleolar and cytoplasmic RNA density during "cell dedifferentiation" represented by blastic transformation of human mature T lymphocytes - a cytochemical study.

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On the nucleolar and cytoplasmic RNA density during "cell dedifferentiation" represented by blastic transformation of human mature T lymphocytes - a cytochemical study.

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