- Email: [email protected]
Image analysis of feulgen-stained ras -transformed NIH 3T3 cell clones
174
Cell Biology
IMAGE ANALYSIS OF FEULGEN-STAINED ras-TRANSFORMED NIH 3T3 CELLCLONES Maria Lsello, Ann F Chambers*. Dept of Cell Biology,IB,UNICAMP, Campinas, Brazil; London, *London Regional Cancer Centre, Ontario, Canada DNA amounts and nuclear phenotypes were studied byimageanalysisinFeulgen-stained preparations of different clonesofNIH 3T3 cells, Dreviouslv transformed with the T24 H-ras'oncogene. ill of the ras-transformed celllinesweretumoricrenic.butthevdiffered in terms of their ~21 expression -and experimentalmetastatic ability.The objective was to determine the degree of chromatin condensation in these cell lines.In all of the ras-transformed cell lines,themajority of the nuclei exhibited a DhenOtVDe characterized by the presence02 increasedareas of condensed chromatin, a phenotype not observed in NIH 3T3 cells. All cell lines were heterogeneous for frequency of nuclei with this phenotype(w92%ina clone that is poorly metastatic and expressesverylittle p21;~63% ina poolofhighlymetasta
P381
MORPHO-FUNCTIONAL STUDY OF 3LL LUNG .PR8.1 --METASTASIS VASCULARIZATION Bnlique Hihi% Ehilia Bodeflo, Joau sim6n, Manuel GadaSam, Salvador F. Aliito. Dept. Biologfa Celular y Ciencias Morfolticas. Fat. Me&h. Universidad de1 Pafs Vasco. Leioa. 4WO Vizcaya. Spain. Tumor blood vessels are indispensable for tumor grow& The knowkdge of the morphology and the circulatory function of tumor vessels is important for diagnosis and cancer therapy. In the present work, we have studied the vaseulatnre of expedimental lung metastasis after intravenous injection of Lewis Lung carcinoma cells to syngeneic CS7BU6 mice. Animsls were sac&iced at times ranging from 7 to 21 days after tumor cell injection. Multiple lung pieces, less than 1 mm2. were nrocessed for transmission elWtron micmcopy, by c&vent&l metbods. Punctionsl vasculamre was “in vivo” studied by means of the Hoechst 33342 fluorescent DNA staining technique. Thus, lung metastases were tnc&ced as above described and, 1 or 10 min before sacrifk~ mice were iujected with 0.25 ml of a solution of Hoe&t 33342 (1 mg/ml concentrationinsterilesaline)intoa lateral tail vein. Subsequently, lungs were fured in liquid nilrogen, and frozen sections studied and photographed under ultraviolet light. Tbe results showed a neoplasm composed of large cells arranged in sheets with a thin irregularly distributed stroma. Scattaed blood vessels with an open or closed lumen were observed within the tumor focus. Stroma around blood vessels was observed as a thick basal lamina. Functional study of Hoechst 33342 diffusion showed that narenchvmal metastatic foci displayed a reticular fluoresce& pat&. In contrast, metastases located around blood vessels and conducting airways did net displayed fluorescence. In summary, our results suggest that the intmorgan location modulates the characteristics of metabolic exchange of the tumor cells in relation to the vascular organization of the mctastatic focus. This work was supported by grant UPV 07X327-16/89 from the University of Pafs Vasco.
International
Reports,
Vol. 74, Abstracts
Supplement
1990
DNA CONTENT AND CHROMATIN STRUCTURE IN HUMAN RENAL CARCINOMA (HRC) Dept of Cell Benedict0 de Campos Vidal. Biology, Institute of Biology, UNICAMP, Campinas (SP), Brazil DNA amount variation and the chromatin packing state were determined by scanning microspectrophotometry in Feulgen-stained Chicken preparations from 5 HRC cases. erythrocyte nuclei (DNA, 2pg) and normal human kidney cells (DNA, 6.5pg) were used DNA as controls. All HRC cases depicted content variations. Micronuclei were aetected with about 2 DNA pg. A nuclear population exhibiting 4-5 DNA pg could be the “stem cell" of the aneuploid and PolYploia cell population of the HRCs. The correlation of Feulgen-stained nuclear areas on the Feulgen-DNA values was positive for all the cases studied (r= 0.65, p < 0.01). An increase in nuclear area was found in the HRC as compared to thecontroL As a consequence, the HRC nuclei showed a decrease in area covered by condensed chromatin (CC) as defined by an absorbance value, A = 0.450 (cut off point), above which thgfcondensation was distinguished . the average absorption ratio In addition, (AAR = Acf/At) of HRC was higher than that of the control (1.9x). This means that the tumor cell nuclei had a higher area and deconexhibited a more intense chromatin densation, but their remained CC was more condensed than was that of the control. Supported by FAPIUNICAMP, FAPESP
P382
Joop ten Kate, Hiaayoahi Sate*, WIlland N.M. Dh@s, Peter T.M. Mocrkerk, Edith P.M. van dcx Linden, Pred T. Bosmau. Department of Pathology, Unksity of Lbbuq. P.O. Box 616, 6200 MD -.m N -*PrcaQntsddress: II, F%lknhM Medical college, l3ikdm& A regcmeratiug liver may form a better “soil” for colon tumor cells tbaaanormalliver.Totcsttbiahypotk&wedidthefollr&ng wpcrimentPartial~alldhslflobectomywerepcrfomlcd iaBalb/cmice.Aftat48ami%lumrsape&iuthelab&&du oftherqeneratins liv8rwasoberwd.Inthehalfbbectomymice
cxpr~3~af~6~d72houraWithinsih1 hytaSationwecouldvkuakcthelocaliacreaseofmycinthe halt lobectomised lobe. The optimal time to iutroduce tumor cells in the regenerating liver wasfamdtobeiIladdyaltcr~beualse~’ src wpposcd to be optimal in that period. From a aeric3 of 30 nude mice 10 were partial (Z/3) hepat&10 half lobcctomkd and1owerenottreated.Intllepartialhepat&omisedauimals5 spleMliaj~ofcolon out of 10 animala allowed metamesafter tumor&(~Lsl74T).Afterbalf~4outoflOahowed metastascsmaialyinthchalfl~lobcandinthccontrol group only two animals with metaatases wwe observed. So we conclnde that: Hepatectomy is a good model to get an increased number 1. of metaatases. 2. Half lobectomy also increase8 the intidence of metastaces, which might have implications for the clinic.
Cell Biology
IMAGE ANALYSIS OF FEULGEN-STAINED ras-TRANSFORMED NIH 3T3 CELLCLONES Maria Lsello, Ann F Chambers*. Dept of Cell Biology,IB,UNICAMP, Campinas, Brazil; London, *London Regional Cancer Centre, Ontario, Canada DNA amounts and nuclear phenotypes were studied byimageanalysisinFeulgen-stained preparations of different clonesofNIH 3T3 cells, Dreviouslv transformed with the T24 H-ras'oncogene. ill of the ras-transformed celllinesweretumoricrenic.butthevdiffered in terms of their ~21 expression -and experimentalmetastatic ability.The objective was to determine the degree of chromatin condensation in these cell lines.In all of the ras-transformed cell lines,themajority of the nuclei exhibited a DhenOtVDe characterized by the presence02 increasedareas of condensed chromatin, a phenotype not observed in NIH 3T3 cells. All cell lines were heterogeneous for frequency of nuclei with this phenotype(w92%ina clone that is poorly metastatic and expressesverylittle p21;~63% ina poolofhighlymetasta
P381
MORPHO-FUNCTIONAL STUDY OF 3LL LUNG .PR8.1 --METASTASIS VASCULARIZATION Bnlique Hihi% Ehilia Bodeflo, Joau sim6n, Manuel GadaSam, Salvador F. Aliito. Dept. Biologfa Celular y Ciencias Morfolticas. Fat. Me&h. Universidad de1 Pafs Vasco. Leioa. 4WO Vizcaya. Spain. Tumor blood vessels are indispensable for tumor grow& The knowkdge of the morphology and the circulatory function of tumor vessels is important for diagnosis and cancer therapy. In the present work, we have studied the vaseulatnre of expedimental lung metastasis after intravenous injection of Lewis Lung carcinoma cells to syngeneic CS7BU6 mice. Animsls were sac&iced at times ranging from 7 to 21 days after tumor cell injection. Multiple lung pieces, less than 1 mm2. were nrocessed for transmission elWtron micmcopy, by c&vent&l metbods. Punctionsl vasculamre was “in vivo” studied by means of the Hoechst 33342 fluorescent DNA staining technique. Thus, lung metastases were tnc&ced as above described and, 1 or 10 min before sacrifk~ mice were iujected with 0.25 ml of a solution of Hoe&t 33342 (1 mg/ml concentrationinsterilesaline)intoa lateral tail vein. Subsequently, lungs were fured in liquid nilrogen, and frozen sections studied and photographed under ultraviolet light. Tbe results showed a neoplasm composed of large cells arranged in sheets with a thin irregularly distributed stroma. Scattaed blood vessels with an open or closed lumen were observed within the tumor focus. Stroma around blood vessels was observed as a thick basal lamina. Functional study of Hoechst 33342 diffusion showed that narenchvmal metastatic foci displayed a reticular fluoresce& pat&. In contrast, metastases located around blood vessels and conducting airways did net displayed fluorescence. In summary, our results suggest that the intmorgan location modulates the characteristics of metabolic exchange of the tumor cells in relation to the vascular organization of the mctastatic focus. This work was supported by grant UPV 07X327-16/89 from the University of Pafs Vasco.
International
Reports,
Vol. 74, Abstracts
Supplement
1990
DNA CONTENT AND CHROMATIN STRUCTURE IN HUMAN RENAL CARCINOMA (HRC) Dept of Cell Benedict0 de Campos Vidal. Biology, Institute of Biology, UNICAMP, Campinas (SP), Brazil DNA amount variation and the chromatin packing state were determined by scanning microspectrophotometry in Feulgen-stained Chicken preparations from 5 HRC cases. erythrocyte nuclei (DNA, 2pg) and normal human kidney cells (DNA, 6.5pg) were used DNA as controls. All HRC cases depicted content variations. Micronuclei were aetected with about 2 DNA pg. A nuclear population exhibiting 4-5 DNA pg could be the “stem cell" of the aneuploid and PolYploia cell population of the HRCs. The correlation of Feulgen-stained nuclear areas on the Feulgen-DNA values was positive for all the cases studied (r= 0.65, p < 0.01). An increase in nuclear area was found in the HRC as compared to thecontroL As a consequence, the HRC nuclei showed a decrease in area covered by condensed chromatin (CC) as defined by an absorbance value, A = 0.450 (cut off point), above which thgfcondensation was distinguished . the average absorption ratio In addition, (AAR = Acf/At) of HRC was higher than that of the control (1.9x). This means that the tumor cell nuclei had a higher area and deconexhibited a more intense chromatin densation, but their remained CC was more condensed than was that of the control. Supported by FAPIUNICAMP, FAPESP
P382
Joop ten Kate, Hiaayoahi Sate*, WIlland N.M. Dh@s, Peter T.M. Mocrkerk, Edith P.M. van dcx Linden, Pred T. Bosmau. Department of Pathology, Unksity of Lbbuq. P.O. Box 616, 6200 MD -.m N -*PrcaQntsddress: II, F%lknhM Medical college, l3ikdm& A regcmeratiug liver may form a better “soil” for colon tumor cells tbaaanormalliver.Totcsttbiahypotk&wedidthefollr&ng wpcrimentPartial~alldhslflobectomywerepcrfomlcd iaBalb/cmice.Aftat48ami%lumrsape&iuthelab&&du oftherqeneratins liv8rwasoberwd.Inthehalfbbectomymice
cxpr~3~af~6~d72houraWithinsih1 hytaSationwecouldvkuakcthelocaliacreaseofmycinthe halt lobectomised lobe. The optimal time to iutroduce tumor cells in the regenerating liver wasfamdtobeiIladdyaltcr~beualse~’ src wpposcd to be optimal in that period. From a aeric3 of 30 nude mice 10 were partial (Z/3) hepat&10 half lobcctomkd and1owerenottreated.Intllepartialhepat&omisedauimals5 spleMliaj~ofcolon out of 10 animala allowed metamesafter tumor&(~Lsl74T).Afterbalf~4outoflOahowed metastascsmaialyinthchalfl~lobcandinthccontrol group only two animals with metaatases wwe observed. So we conclnde that: Hepatectomy is a good model to get an increased number 1. of metaatases. 2. Half lobectomy also increase8 the intidence of metastaces, which might have implications for the clinic.