Harvey M. Patt

A CHARACTERIZATION OF THE BOUNDARY BETWEEN THE CYCLING AND RESTING STATES IN ASCITES TUMOR CELLS

Growth deceleration of an Ehrlich ascites tumor with increasing mass is associated with a prolongation of the cell cycle and a decline in the growth fraction. These effects are reversed upon transfer of cells from an older tumor into a new host. Studies were made to locate the stages at which a cell cycle could be suspended or resumed. Transplantation caused a prompt rise in both mitotic and flash H3TdR labeling indices. When all the cells in cycle including mitoses were prelabeled with H3TdR in older tumors, the fraction of labeled mitoses did not decline for a considerable period after transplantation into new hosts. This suggests that the early rise in mitoses is not due to a flow of resting (Go) cells from a G2 store (G2‐Go transition). It appears rather to be a reflection of a lag of the mitotic process relative to other stages during the initial readjustment of the cycle. A prompt rise in flash H3TdR indices in the transplants suggested cell entry into S from either a suspended GI (G1‐Go transition) or a suspended S (S‐Go transition). These possibilities were examined by relating micro‐spectrophotometric estimates of DNA to the cell cycle stage as revealed by H3TdR autoradiography. Since Go cells had DNA values corresponding to GI, it was concluded that decycling or recycling could occur only after mitosis and before DNA synthesis.

MEASUREMENT OF DNA-SYNTHESIS TIME IN MYELOID-ERYTHROID PRECURSORS.

Abstract A technique of double labeling with 3 H-thymidine in vivo followed by 14 C-thymidine in vitro was developed to measure the in vivo period of DNA-synthesis for myeloid-erythroid precursors in the marrow of dogs, one hematologically normal man and one case of acute myeloid leukemia. Results were in the approximate range of 5–6 hr. A completely in vitro measurement with 3 H-thymidine followed by 14 C-thymidine and vice versa showed a three-fold prolongation of the synthesis time in canine myelocytes and erythroid precursors.

Bone Formation and Resorption as a Requirement for Marrow Development

Summary The formation of a new sinusoidal marrow in a heterotopic autologous marrow implant requires the intermediation of bone. This conclusion follows from the intimate association of developing marrow tissue and newly formed bone in the implantation site. It has been shown that there is a sigmoidal relationship between the evolution of a medullary cavity and ossicle volume. Moreover, the mean relative medullary cavity size and ossicle volume for each stage of marrow development from hemocytoblasts to a sinusoidal structure conforms to this relationship. Hematopoietic foci emerge during the period of rapid bone resorption. A fully developed marrow may occur in a small or large ossicle provided that the medullary cavity represents a critical fraction of the total volume. It is suggested that processes of bone resorption may contribute to the formation of an appropriate microenvironment and/or provide a source of appropriately modulated progenitor cells. Significantly, bone plays a necessary role in the genesis of an organized marrow in an evacuated medullary cavity as well as in a heterotopic marrow implant. The authors thank Miss G. Dunn and Mrs. M. Miller for technical assistance.

ORIGIN OF REPOPULATING CELLS AFTER LOCALIZED BONE MARROW DEPLETION.

The restoration of marrow in a mechanically depleted segment of rabbit femur is locally determined and apparently initiated by cells normally resident in bone. This conclusion follows from results of two types of radiation experiments: local x-irradiation of the femur shaft immediately before depopulation and x-irradiation of the total body with exception of the femur diaphysis which was depopulated either just before or after irradiation. In contrast to the rapid initiation of marrow restoration in an unirradiated femur, there was little regeneration during the first 3 weeks in an irradiated femur. Recovery of the shielded depopulated femur in the rabbit that otherwise received total-body irradiation was similar to that in the unirradiated animal. Hence, it would appear that the seeding of circulating hemic stem cells is not essential to repopulation and that cells with hematopoietic potential are present in osseous tissue.

Non-protein sulfhydryl content and cell-cycle dynamics of Ehrlich ascites tumor

Abstract The non-protein sulfhydryl (NPSH) content of Ehrlich ascites tumor cells was measured in relation to cytokinetic parameters of tumor growth. Thymidine labeling index, NPSH, mitotic index, and protein were assayed under a variety of experimental conditions, both in the hours following transplantation and during subsequent daily growth of the tumor. In some experiments, X-radiation was used to block cells in G2. Cellular NPSH declines during progressive tumor growth. This decrease appears to reflect the increasing proportion of non-proliferating (G0 or G1-like) cells or the decreasing proportion of proliferating cells in the tumor. When cells from 7-day tumors are transplanted into fresh mice, G0 cells move back into the cycle at S and the mean cellular NPSH content more than doubles within 9 h. When large inocula are used, the post-transplantation increases in the proportion of cells synthesizing DNA and in the mean NPSH level are greatly reduced. These results suggest that G0 cells contain less NPSH than do proliferating cells. There is no evidence that proliferating cells contain more NPSH in late G2 or in mitosis than in any other phase of the cell cycle. The rise in cellular NPSH concentration following transplantation was not affected by prior X-radiation (1250 R), indicating that movement of G0 cells back into the cell cycle is not particularly radiosensitive.

Increased survival of haemopoietic pluripotent stem cells in vitro induced by a marrow fibroblast factor.

Summary. When mouse bone marrow was incubated in medium conditioned by marrow fibroblasts, the survival of pluripotent stem cells (CFUS) was considerably greater than when marrow was incubated in fresh medium. This increase in CFUS survival depended on the age of the marrow fibroblast culture, the initial number of cells in the culture, and the concentration of the conditioned medium. Medium conditioned by fibroblasts from other adult tissues—spleen, bone, and subcutaneous tissue—did not increase CFUS survival, but medium conditioned by embryo bone did. The increase in CFUS survival by marrow‐fibroblast‐conditioned medium was not accompanied by any change in the total number of nucleated cells of the incubated marrow nor by any comparable increase in the survival of granulopoietic stem cells (CFUC) or erythropoietic stem cells (BFUE). These results indicate that marrow fibroblasts produce a factor that increases the survival of CFUS, which may be involved in the role of marrow stroma in the control of haemopoiesis.

Relationship of bone marrow cellularity and proliferative activity: a local regulatory mechanism.

An analysis has been made of tritiated thymidine (3H‐TdR) uptake and cellularity in normal and regenerating marrow. Comparison of right and left femoral marrow in ninety normal rabbits has revealed a significant negative correlation (−0.75) between thymidine uptake per presumptive proliferative cell and the concentration of such cells. Locally irradiated marrow follows the normal marrow regression down to about 40% relative cellularity and then shows an upward displacement. The regenerating mechanically depopulated marrow manifests a similar displacement over the entire cellularity range studied which suggests that recovery was not yet complete as in the case of some of the irradiated marrow samples. The negative regression of 3H‐TdR uptake on cellularity is not a consequence of altered thymidine availability, but rather of‐a changing DNA synthesis rate and/or a changing fraction of proliferative cells. Since 3H‐TdR uptake per presumptive proliferative cell reflects cell production, it follows that cell production is geared to the cellularity of a discrete marrow area. This provides a local self‐regulating mechanism.

Evolution of marrow regeneration as revealed by transplantation studies

Abstract This study is concerned with the regeneration of bone marrow as an organized tissue. It is addressed specifically to the time in the regenerative program when the emerging tissue acquires the potential to reconstitute marrow. The evolution of this potential was investigated in rabbits by making autologous subcutaneous implants of tissue obtained at intervals after evacuation of a femur shaft. Analysis of 140 implant sites (89 of regenerating tissue and 51 of normal marrow) reveals a striking similarity in development of implants of a 2 to 4 day regenerating tissue and of normal marrow. New marrow encapsulated by bone can be seen in each instance 5 weeks after implantation. This property of regenerating tissue is uncovered before there are any obvious hemic elements. Significantly, the likelihood of a take is greatly increased when the implant contains a connective tissue. Marrow was always found in the implantation site when an ossicle was forming. It never occurred in the absence of bone. We conclude from this study that the appearance of modulated mesenchymal elements in the early regenerating tissue imparts the quality of normal marrow.

GRANULOCYTE DYNAMICS AND THE QUESTION OF INEFFECTIVE GRANULOPOIESIS

A simple analysis was developed to compare granulocyte production in marrow with granulocyte turnover in peripheral blood of the dog. the analysis is anchored to the relative number of granulopoietic and erythropoietic cells that are flashlabeled with tritiated thymidine. This provides a convenient measure of relative production rates, since there is little, if any, difference in the duration of the respective DNA synthesis periods. the latter was confirmed in the present work by comparison of myeloid and erythroid cells in respect to (1) labeled mitosis curves and (2) changes in labeling intensity with constant infusion of tritiated thymidine. the ratio of flash‐labeled granulocyte to erythrocyte progenitors was unity (1.02 ± 0.05) in the eight dogs studied, which means that gross production rates were essentially the same. Since net production as revealed by peripheral blood turnover is greater for erythrocytes than for granulocytes, it follows that there must be some ineffective granulopoiesis. This may correspond to over half of the total production. It is suggested that this phenomenon, which apparently occurs in man as well as in dog, could represent an important feature of granulocyte regulation.

The Question of Bone Marrow Stromal Fibroblast Traffic

Bone marrow stromal fibroblasts (CFU-F) normally do not exchange bone marrow sites in vivo. Restitution of the CFU-F after radiation damage is primarily recovery by the local fibroblasts from potentially lethal damage. Migration of stromal fibroblasts from shielded sites to an irradiated site makes a minimal contribution, if any, to CFU-F recovery. Determination of the relative contribution of donor stromal cells in bone marrow transplants by karyotyping the proliferating bone marrow stromal cells in vitro may not reflect the relative distribution of fibroblasts in the marrow. If there is residual damage to the host stromal fibroblasts from treatment before transplantation, these cells may not be able to proliferate in vitro. Therefore, an occasional transplanted fibroblast may contribute most of the metaphase figures scored for karyotype.