RESUMO
To determine whether all tumor S-phase cells incorporate [3H]thymidine, we labeled the cells in three mouse tumors (MCa-11, colon-26, and colon-51) in vivo for 1/2 h with [3H]thymidine (10 microCi/g of body weight). Cells from the tumors, as well as control cells from the bone marrows of the tumor-bearing mice, were then placed onto slides and Feulgen stained. The positions of these Feulgen-stained cells were mapped with a computerized scanning stage, and their nuclear DNA content and nuclear areas were determined by absorption cytophotometry. Next, the slides were processed for autoradiography and exposed for 32 or 64 days to obtain plateau labeling. The cells were then relocated, and the areas of the autoradiographic grains over each nucleus were measured. We found that 99% of the S-phase bone marrow cells were labeled. The 5-mm tumors, however, showed a wide range of S-phase labeling, with 94, 89, and 85% of the MCa-11, colon-51, and colon-26 S-phase cells, respectively, being labeled. The same mice bearing both 5- and 20-mm MCa-11 tumors, however, showed 95 and 57% labeling of the S-phase cells in the small and large tumors, respectively. These results show that the [3H]thymidine labeling of S-phase cells varies greatly for experimental mouse tumors of different size and type, and they suggest that "labeling index" and flow cytometric DNA measurements may not give valid estimates of the actual proportions of cycling S-phase cells in such tumors.
Assuntos
DNA de Neoplasias/biossíntese , Interfase , Neoplasias Experimentais/patologia , Animais , Medula Óssea/patologia , Divisão Celular , Núcleo Celular/ultraestrutura , Replicação do DNA , CamundongosRESUMO
Mouse mammary carcinoma (MCa-11) cells were grown in vitro in exponential, plateau-fed, and starved monolayer cultures or as 100-, 250-, and 500-microns tissue culture spheroids, and in vivo as small (4-mm diameter) and large (12-mm diameter) tumors. In all of these forms, the growth rates of the MCa-11 cells were found to decrease after an initial rapid proliferation of a relatively small number of cells. The DNA distributions of these cells during different rates of growth in vitro and in vivo, as well as the proportion and intensity of labeling of the S-phase cells with [3H]thymidine and [3H]deoxyuridine, were measured by flow and absorption cytometry. We found that significant numbers of MCa-11 cells remained in S phase, even after the growth rates in vivo and in vitro had slowed. However, as growth rates decreased, the intensity and proportion of S-phase cells labeled with exogenous DNA precursors decreased. We conclude that progressive alterations, including possible slowing and cessation, of replicative DNA synthesis occur in S-phase tumor cells as the metabolic constraints on tumor growth are increased.
Assuntos
DNA de Neoplasias/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Animais , Células Cultivadas , DNA de Neoplasias/análise , Desoxiuridina/metabolismo , Feminino , Interfase , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Timidina/metabolismo , TrítioRESUMO
A computerized "flying spot" microdensitometer and scanning stage have been combined to measure the cellular DNA content, nuclear areas, and autoradiographic grain areas of the same cells. The slide positions of the Feulgen-stained, (3H)thymidine-labeled cells are mapped with the computerized stage, and nuclear DNA content and areas are then determined by integral absorbance measurements at 588 nm. Following autoradiograph preparation, the cells are relocated and the areas of the autoradiographic grains over each nucleus are measured at a light wavelength (625 nm) and an optical density setting (greater than 0.10) that do not detect the Feulgen stain. The microcomputer calculates the portion of each nucleus covered with autoradiographic grains (grain area proportion, GAP), and it links the GAP value to the DNA content of each nucleus in the computer file for subsequent sorting and analysis. By using this system in a study of mouse bone marrow cells labeled in vivo with (3H)thymidine, we found that all S-phase cells were clearly labeled after 8 or more days of autoradiographic exposure. Prolonged exposures (up to 64 days) led to detection of lightly labeled cells (0.1 less than GAP less than 0.8) with G1/G0 and G2 DNA content.
Assuntos
Medula Óssea/metabolismo , Núcleo Celular/metabolismo , DNA/metabolismo , Animais , Autorradiografia , Medula Óssea/ultraestrutura , Células da Medula Óssea , Núcleo Celular/ultraestrutura , Computadores , Técnicas Citológicas/instrumentação , Interfase , Camundongos , Timidina , Fatores de Tempo , Distribuição Tecidual , TrítioRESUMO
A method of improving absorption cytophotometric cellular DNA values by making measurements on Feulgenstained cells at optimal stain absorbances has been developed. Stain intensity can be controlled either by alteration of the Feulgen staining reaction or by selection of "off-peak" wavelengths of light for cytometry. The use of chicken red blood cells as an internal standard, and of a computerized cytometer for the measurements, allows selection of the appropriate off-peak light wavelengths, correction for staining variability at different sites on the same slide, and rapid calculation of cellular DNA values. Cytometry can also be performed at controlled absorbance levels on autoradiographs of 3H-thymidine-labeled cells to allow direct study of the DNA content of nonlabeled G1/G0 and G2/M cells. Use of this technique on mixtures of mouse thymocytes, spleen cells, bone marrow cells, and liver cells gave essentially identical values for G1/G0 cellular DNA content, with coefficients of variation of less than 3%.
Assuntos
DNA/análise , Eritrócitos/análise , Corantes de Rosanilina , Linfócitos T/análise , Animais , Autorradiografia , Galinhas , Corantes , Camundongos , Fotometria , Coloração e Rotulagem/métodosRESUMO
We report on a study of the cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mice of the C57B1/6J (with high-affinity TCDD receptor) or DBA/2J (with low-affinity TCDD receptor) strains were given single intraperitoneal injections of 50, 100 or 150 micrograms of TCDD/kg body weight. At various times (8-48 h) after injection, we examined bone marrow cells for cytogenetic effects by performing structural aberration, sister-chromatid exchange, and micronucleus tests. 1 month after exposure, liver sections were studied for hepatotoxic effects. We found no evidence of chromosome damage by TCDD given in doses that cause liver damage in both strains of mice.
Assuntos
Medula Óssea/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Dioxinas/toxicidade , Fígado/patologia , Mutagênicos , Mutação , Dibenzodioxinas Policloradas/toxicidade , Animais , Células da Medula Óssea , Aberrações Cromossômicas , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Testes de Mutagenicidade/métodos , Troca de Cromátide Irmã/efeitos dos fármacosRESUMO
Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [3H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G0/G1, S, and G2/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [3H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [3H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G2 tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.
Assuntos
Ciclo Celular , Citofotometria , DNA de Neoplasias/análise , Citometria de Fluxo , Neoplasias Mamárias Experimentais/análise , Absorção , Animais , Interfase , Cinética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neoplásicas/análise , Timidina/metabolismo , Trítio , Células Tumorais CultivadasRESUMO
Spheroids from the V279-171b and MCa-11 cell lines were incubated continuously for 24 hr in [3H]thymidine for labelling of the outer cells of the viable rim. The spheroids were dispersed into single cells, and the DNA content of photomapped cells was measured by absorption cytophotometry. Autoradiographs were then prepared from which we ascertained cellular labelling. For spheroids of both cell lines, we found a larger proportion of cells with a G0/G1 DNA content among the non-labelled inner spheroid cells than among the labelled outer cells (P less than 0.001). This block of non-labelled spheroid cells in G0/G1 was not a cell cycle perturbation caused by the isotope for the MCa-11 spheroids. Approximately 8% of non labelled MCa-11 spheroid cells had S/G2 DNA content, suggesting that non-cycling cells in spheroids may be blocked in S and G2 as well as in the G0/G1 phase of the cell cycle.
Assuntos
Células Cultivadas/citologia , DNA/análise , Interfase , Animais , Autorradiografia , Linhagem Celular , Separação Celular , Células Cultivadas/análise , Cricetinae , Metáfase , Camundongos , Espectrofotometria , Timidina/metabolismoRESUMO
We have developed a method of calculating the average local absorbance (ALA) of a nucleus from the integrated nuclear absorbance and area. One can use the ALA, along with nuclear areas measured at different point absorbance thresholds, to determine whether a nucleus is stained too lightly or too darkly for accurate absorption measurements; this allows selection of an optimal light wavelength for the performance of these measurements. The ALA can also be used for automatic and instantaneous correction of integrated absorbance values from darkly stained cells. This allows the rapid measurement of the integrated absorbances of a large number of nuclei that are heterogeneous in stain intensity. Coefficients of variation of approximately 3% are obtained for the integrated absorbances of nuclei of nontransformed G0/G1 cells. This correction method can be applied with any image densitometer that generates both integrated absorbance and area values.