Initial Labeling Index Research Articles

P53 and MIB-1 immunohistochemistry as predictors of the clinical behavior of nonfunctioning pituitary adenomas.

P53 expression and increased MIB-1 proliferation index have been shown to correlate with invasive behavior in pituitary adenomas. The purpose of this study was to determine whether these indices could be used to predict a higher likelihood of recurrence in clinically nonfunctional pituitary adenomas and thus guide adjuvant therapy. Fifty-one clinically nonfunctional pituitary adenomas were selected from the database at the Vancouver Hospital and Health Sciences Center between the years 1990-1998. Included were 32 nonrecurrent and 19 recurrent adenomas. The mean initial labelling index for p53 in nonrecurrent tumours was 0.38% (0-1.58%), while it was 0.46% (0-3.65%) for recurrent adenomas. The mean initial MIB-1 index for nonrecurrent tumours was 1.63% (0.08-9.36%), while for recurrent tumours it was 1.92% (0-7.76%). The percentage of p53 positive adenomas was 66% for nonrecurrent tumours and 68% for recurrent tumours. None of the differences in the labelling indices between the recurrent and nonrecurrent groups was statistically significant. As 12 patients (38%) in the nonrecurrent group had undergone radiotherapy as initial adjuvant therapy after surgery and none of the recurrent group had done so, patients who did not receive radiotherapy in the nonrecurrent group were analyzed separately. Again, none of the differences in the labelling indices between the recurrent and nonrecurrent groups was statistically significant when the effect of radiotherapy was removed from the analysis. The results demonstrate no statistical difference in the p53 or MIB-1 labelling indices between recurrent and nonrecurrent nonfunctional pituitary adenomas. Concern should be raised in attaching too much clinical significance to these labelling indices, especially with respect to p53 as a predictor of the clinical behavior of nonfunctional pituitary adenomas.

Megakaryocy topoiesis: Incorporation of Tritiated Thymidine by Small Acetylcholinesterase-Positive Cells in Murine Bone Marrow During Antibody-Induced Thrombocytopenia

The ability of small acetylcholinesterase-positive (SACHE) cells to incorporate tritiated thymidine (3H-TdR) was studied in the bone marrow of mice made acutely thrombocytopenic by injection of guinea pig antimouse platelet serum (APS) and in control mice injected with normal guinea pig serum (NS). 3H-TdR was administered in vivo, and bone marrow was collected at various time points thereafter. The initial labeling index (LI) for both groups was about 30%. After four hours, the LI increased and reached peak values at 48 hours, but was lower in thrombocytopenic animals. The lower peak LI in APS-treated animals may be the result of both a faster rate of influx of unlabeled cells into the acetylcholinesterase (AChE) positive cell compartment and efflux of more heavily labeled, and probably polyploid, SACHE cells into the compartment of recognizable megakaryocytes. In both groups the increasing LI occurred concomitantly with a decreasing mean grain count. This may indicate cellular division by some fraction of SACHE cells. Heterogeneity in nuclear morphology was also demonstrated, and the frequency of individual morphologies was altered in APS-treated animals. In addition, cells that incorporated 3H-TdR were larger than those that did not. This group of small cells appears to represent a pivotal point in megakaryocytopoiesis in which the switch from mitosis to endomitosis is occurring.

Megakaryocytopoiesis: incorporation of tritiated thymidine by small acetylcholinesterase-positive cells in murine bone marrow during antibody-induced thrombocytopenia

The ability of small acetylcholinesterase-positive (SACHE) cells to incorporate tritiated thymidine (3H-TdR) was studied in the bone marrow of mice made acutely thrombocytopenic by injection of guinea pig antimouse platelet serum (APS) and in control mice injected with normal guinea pig serum (NS). 3H-TdR was administered in vivo, and bone marrow was collected at various time points thereafter. The initial labeling index (LI) for both groups was about 30%. After four hours, the LI increased and reached peak values at 48 hours, but was lower in thrombocytopenic animals. The lower peak LI in APS-treated animals may be the result of both a faster rate of influx of unlabeled cells into the acetylcholinesterase (AChE) positive cell compartment and efflux of more heavily labeled, and probably polyploid, SACHE cells into the compartment of recognizable megakaryocytes. In both groups the increasing LI occurred concomitantly with a decreasing mean grain count. This may indicate cellular division by some fraction of SACHE cells. Heterogeneity in nuclear morphology was also demonstrated, and the frequency of individual morphologies was altered in APS-treated animals. In addition, cells that incorporated 3H-TdR were larger than those that did not. This group of small cells appears to represent a pivotal point in megakaryocytopoiesis in which the switch from mitosis to endomitosis is occurring.

Evidence of Mouse Epidermal Subpopulations with Different Cell Cycle Times

In order to obtain information on the distribution of total cell cycle times in hairless mouse epidermis, basal cells were isolated and prepared for DNA flow cytometry at intervals after a pulse labeling with 50 microCi of thymidine. The DNA distributions were recorded, and cells were sorted from windows in the S, G2, and G1 phases of the cell cycle, collected on glass slides, and subjected to autoradiography. The proportions of labeled cells were scored in each fraction, and the percentage of labeled mitoses was determined in histologic sections from the same animals. Grain count distributions were recorded at selected time points over labeled cells in sorted fractions and over labeled mitoses. The movement of the labeled S-phase cohort was thus followed through all cell cycle phases. Peaks in labeled cells were observed at about 36 h in S phase, G2 phase, and mitosis, and high levels of labeled G2 cells and mitoses were seen at about 80 h. These results indicate the existence of one rapidly cycling subpopulation of keratinocytes with a cell cycle time slightly less than 30 h, in addition to keratinocytes with considerably longer cell cycle times. The first peak of labeled G2 cells reached only about 30%. This is consistent with earlier findings of about 30% G2 cells with a rapid traverse, and 70% with a considerably delayed traverse through G2 phase. The proportion of labeled G1 cells reached a value corresponding to twice the initial labeling index at 8 h after pulse labeling. This is consistent with previously obtained phase durations, indicating an unperturbed cell cycle traverse of labeled cells from S phase through G2 and mitosis.

Proliferation of human lymphoid cells. Lymphocytopoiesis and cell cycle parameters in isolated perfused human spleens.

Parameters of the cell cycle of lymphoid cells and the rate of production of small lymphocytes and mature plasma cells were estimated in human spleens. 12 normal and 1 enlarged spleen with lymphatic hyperplasia were labelled either by a pulse or continuously with 3H-TdR-thymidine during normothermic perfusion of the isolated spleens. Biopsies of splenic tissue were taken at different intervals and evaluated autoradiographically. The mean initial labelling index of immunoblasts was 73% and for plasmoblasts 52%. The labelling index for small lymphocytes and mature plasma cells increased initially from 0% to 1%, and up to 25% respectively after 14 h. The duration of the S-phase, as determined by double labelling, was 8.26 h for lymphoid blasts. Data from the percentage labelled mitosis curve resulted in a minimal tG2 of about 1 h and tG2 + tM of about 4 h. The estimated generation time was about 11.5 h. The results found on the hyperplastic spleen were comparable to those on the normal spleen.

Proliferation kinetics of malignant non-Hodgkin's lymphomas related to histopathology of lymph node biopsies.

Since lymphomas of high and low malignancy have been differentiated by the Kiel-classification, the cell kinetics of malignant lymphomas have become an interesting subject. In 20 non-Hodgkin's lymphomas we have determined the initial labelling index, the mitosis index, the proliferation rate and the durations of DNA-synthesis and mitosis. The kinetic variables have been estimated by using an in vitro-incubation of fresh biopsies and double-labelling with radioactive DNA-precursors. in 8 lymphomas of low malignancy we found an initial labelling index of 7.8%, a mitosis index of 0.3% and a potential tumour doubling time of 100.2 h for a cell production rate without regard to cell loss. On the other hand, lymphomas of high malignancy showed a labelling index of 16.7%, a mitosis index of 0.7% and a potential tumour doubling time of 40.5 h. All these values could be differentiated with high statistical confidence. DNA-synthesis time and mitosis time of both lymphoma groups did not show any significant differences. From these findings we obtained confirmation of the cyto-morphological principles used in classifying lymphomas.

Changes in the proliferation rate of mouse epidermis after irradiat-on continuous labelling studies.

The proliferative response of mouse skin to damage caused by X-irradiation has been tested by giving repeated injections of tritiated thymidine and scoring the percentage of labelled cells in high resolution autoradiography. Four, nine and fourteen daily fractions of 300 rads of X-rays were used and labelling commenced 4 days after the last fraction. The epidermis of the upper surface and the sole of the foot were scored separately and were compared with the skin of unirradiated feet. In unirradiated skin the proliferation rate of the basal layer cells is more rapid on the sole than on the upper surface. The cell cycle times deduced from continuous labelling curves were 81 hr and 111 hr respectively and the growth fractions were 97% and85%. After irradiation with small daily doses the homeostatic response to cell killing was slow. More rapid proliferation occurred after nine fractions in the sole, but was not apparent in the skin of the upper surface until fourteen fractions had been given. After fourteen fractions the cell cycle time was about 24 hr on both surfaces and the growth fraction was about 90%. The initial labelling index after a single thymidine injection was a poor measure of proliferation rate. The delay in the time of onset of faster proliferation is similar, both qualitatively and quantitatively, to that measured previously from the additional dose increments needed if large doses were given at different times after the multifraction treatments (Denekamp, 1973).

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.

On the existence of a G 0 -phase in the cell cycle.

ABSTRACTThe model is based on the assumption that the cell cycle contains a Go‐phase which cells leave randomly with a constant probability per unit time, γ. After leaving the Go‐phase, the cells enter the C‐phase which ends with cell division. The C‐phase and its constituent phases, the‘true’G1‐phase, the S‐phase, the G2‐phase and mitosis are assumed to have constant durations of T, T1Ts, T2 and Tm, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle.The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and Ts. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the Go‐phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go‐phase.

Cell proliferation in neuroblastoma

In a patient with generalized neuroblastoma labeling index and median grain-count determinations on tumor cells in mitosis and interphase were performed autoradiographically after pulse-labeling with tritiated thymidine. The tumor-cell samples were removed from different parts of a superficially growing metastasis. The mitotic index was 0·57%. Labeled mitoses data indicated a minimum G 2 time of 1–2 hr and a mean DNA synthesis time of approximately 27–28 hr. Mitotic-time calculations yielded a value of 1·4 hr. The initial labeling index of all neuroblastoma cells in interphase was 11%. Classification of tumor cells according to relative nuclear size permitted to distinguish at least two cell compartments: one of cells with large, initially labeled and one of cells with small, initially unlabeled nuclei. The data are compatible with the assumption that there is a continuous reciprocal exchange of cells between these compartments and that the majority of the tumor cells retain the ability to divide.

Megakaryocytopoiesis in Rats With Special Reference to Polyploidy

Abstract The morphologic stage of maturation, ploidy level, and cell size of individual megakaryocytes from the marrow of normal rats were determined; the initial labeling index of this population of megakaryocytes 30 minutes after injection of 3HTdr was also determined. The results suggested some inferences about the flow of cells through the various ploidy and differentiation compartments during the maturation of megakaryocytes. Most 8N Type-I megakaryocytes endoreduplicate and become 16N Type-I cells, while a few mature to 8N Type-II megakaryocytes. Most 16N I cells mature to 16N II although a few endoreduplicate to become 32N I cells; the 32N I cells mature only to 32N II. The Type-I morphologic stage contains two subcompartments that we have not distinguished morphologically, an endoreduplicating subcompartment and a post-DNA-synthesis subcompartment. The time parameter of the latter ranges from about nine to 19 hours. The time parameter of the former depends on the number of endoreduplications undergone by an individual megakaryocyte. The size of megakaryocytes increases both with increases in ploidy level and with cell differentiation.

AN EVALUATION OF ERYTHROPOIESIS IN THE YOUNG RAT

ABSTRACTEstimates of the cell population kinetic parameters have been obtained for the erythroid cells of the young growing rat using the technique of labelled mitoses and these results have been analysed by a computer programme. The phases of the cell cycle for the proliferating cells have been shown to be of shorter duration than generally reported. Together with the differential cell count and initial labelling index these data have enabled estimates of the growth fraction, birthrate, flow rate, number of divisions and transit time to be determined for each compartment.