A mechanism of intracellular timing and its cooperation with extracellular signals in controlling cell proliferation and differentiation, an amended hypothesis.

Various observations suggest that an intracellular timer is involved in the control of cell proliferation and differentiation that supplements control by extracellular signaling and depends on quantitative relations between cytoplasm and nucleus. To further elucidate the mechanism of this timer, we examined the results of experiments with mice in which cell cycle regulating genes were inactivated: the inactivation of negative cell cycle regulators extends cell proliferation, whereas inactivation of positive regulators decreases cell proliferation. We conclude that this is caused in the former case by shortening of G1 which decreases the cytoplasmic growth rate per cell cycle, whereas in the latter case this rate is increased due to G1 prolongation. This is consistent with our hypothesis according to which the cytoplasmic/nuclear ratio must increase to a certain level to induce end stage differentiation and cell cycle arrest. A new basis of this hypothesis is the fact that end stage differentiation requires large quantities of membranous cytoplasmic structures that the cells are unable to produce de novo. Embryonic cells, however, possess only few of these structures. The only feasible way to multiply these structures is by growing more cytoplasm per cell cycle than needed for a doubling so that successively, the level of the cytoplasmic/nuclear ratio is reached that is required for differentiation. A consequence is that the cytoplasmic growth rate per cell cycle determines the number of amplification divisions. We suggest that the differentiation signal may be triggered when a differentiation-preventing protein (for example Bcl-2) is diluted out by the expansion of cytoplasmic membrane structures, thus simultaneously determining the cell size. The intracellular timer and extracellular signals cooperate in adjusting cell production to the organism's need and in determining when and how the cells respond to extracellular signals or transmit extracellular signals.

Control of cell proliferation by progress in differentiation: clues to mechanisms of aging, cancer causation and therapy.

In multicellular organisms, the control of cell proliferation occurs, in part, by modulating the progress in differentiation. In normal and neoplastic cells, for example, progress towards terminal differentiation concludes cell proliferation, whereas arrest of progress in differentiation causes uncontrolled cell proliferation. Evidence is presented according to which the progress in differentiation depends on an increase in the ratio between mitochondrial differentiation promoting activity and nuclear differentiation preventing activity. This ratio is low in embryonic cells and in stem cells, due to low mitochondrial content, but increases by a rate of mitochondrial multiplication that is larger than a doubling of mitochondrial content per cell cycle. The rate of mitochondrial multiplication, thus, decides on the progress in differentiation and controls the number of amplification divisions between cell determination and terminal differentiation. This rate is modifiable by extracellular signals and cellular defects. Mutations, for example in nuclear genes coding for mitochondrial proteins, are likely to decrease the rate so much that differentiation is arrested with ensuing neoplastic growth. Agents used in differentiation therapy and ionizing radiation overcome this arrest: the cell cycle is sensitive to these agents, but not the mitochondria which multiply during the transitory cell cycle inhibition, thus increasing the differentiation promoting activity. Differentiation arrest can be circumvented also by direct inhibition of nuclear differentiation preventing activity at the level of transcription or translation, whereas corresponding inhibition of mitochondrial differentiation promoting activity prevents differentiation. Accumulation of non-specific genetic damage causes persisting cell cycle prolongation and enhancement of differentiation which, apparently, are involved in senescence. The recent finding of increase in mitochondrial mass prior to release of cytochrome c, induction of differentiation, and apoptosis points to similarities in the initial molecular pathways of differentiation and apoptosis.

Radiation-enhanced differentiation of erythroid progenitor cells and its relation to reproductive cell death.

Terminally differentiated cells usually do not divide and are, thus, reproductively dead. To elucidate the significance of radiation-enhanced differentiation to reproductive cell death, murine erythroid progenitor cells were gamma-irradiated in plasma clot cultures and the development of haemoglobinized clones was studied thereafter. If irradiation occurred when the cells had resumed proliferation, the total numbers of haemoglobinized clones and, in parallel, the numbers of newly haemoglobinized clones were elevated above control levels 6-24 h after 10-30 Gy and 24-48 h after 1 Gy respectively. Thereafter, clone numbers decreased below controls. This decrease was faster with the newly haemoglobinized clones, indicating that both the accumulation of haemoglobinized clones and fast exhaustion of the pool of more primitive precursors in the cultures are due to accelerated differentiation. The haemoglobinized clones appearing after irradiation were reduced in size without indication of direct cell death. We conclude that the reproductive cell death occurring in our system is due to enhancement of differentiation. Enhancement of differentiation is expressed by omission of cell cycles normally passed through by the cell progeny before terminal differentiation is reached. Dependence of differentiation enhancement on the presence of cycling cells at the time of irradiation indicates involvement of growth of essential cytoplasmic constituents during mitotic delay as observed in other cell systems.

Induction of differentiation in erythroleukemic K562 cells by gamma-irradiation.

Various agents have been shown to induce differentiation in neoplastic cells. The present study aimed at investigating comparable phenomena induced by high doses of gamma-irradiation in the presence of physiological factors. The erythroleukemic K562 cells were gamma-irradiated or treated with cytosine-arabinoside (Ara-C), and examined for cell size, protein content, acetylcholinesterase (AChE)-activity and hemoglobin synthesis in relation to mitotic activity. At doses above 10 Gy, differentiation was induced, as recognized by elevated AChE-activity, accompanied by an increase in cell size and protein content and cessation of cell proliferation. Moreover, irradiation, as well as Ara-C, induced hemoglobin synthesis when cultures were supplemented with hemin prior to treatment. It is suggested that the basic mechanisms of differentiation induction are similar for ionizing radiation and certain chemical agents and are related to continued growth of essential cytoplasmic constituents during inhibition of mitotic activity.

Review: a major component of radiation action: interference with intracellular control of differentiation.

If genetic lesions were the sole reason of damage induced by ionizing radiation, an increase in the number of identical chromosome sets (polyploidy) may be expected to have a radioprotective effect. This effect is evident in terminally differentiated tissues when the reduction in remaining life span is used as the criterion. This effect is also evident in cells capable of proliferation if cytoplasmic growth during the period of mitotic delay is restricted and the criterion used is continuation of cell proliferation. Both instances demonstrate that polyploidy, in principle, can exert a radioprotective effect, although the genetic damage induced by a given dose increases in approximate proportion to ploidy. However, in mitotically active cells, without restrictions in cytoplasmic growth, differentiation enhancement dominates the effects of genetic lesions, and polyploidy does not protect. Enhancement of differentiation causes damage by eliminating amplification divisions normally passed through by cell progenies before terminal differentiation, thus reducing the number of differentiated cells produced. From its dependence on excess cytoplasmic growth it is concluded that the phenomenon is caused by the interference of ionizing radiation with a mechanism that provides intracellular signals needed to coordinate molecular interactions involved in the control of cell differentiation. This conclusion corresponds to experiments that suggest that intracellular control of differentiation depends on an increase in the ratio of essential cytoplasmic constituents, probably mitochondrial genomes, per nuclear genome. The action of chemical differentiation enhancing agents is similar and an outline of probable mechanisms is presented. Regarding late radiation damage it is concluded that non-specific genetic lesions can enhance differentiation by permanently prolonging the cell cycle, which causes an increased cytoplasmic growth rate per cycle. In this case polyploidy cannot protect because the induced genetic lesions are proportional to ploidy. Both the duration of mitotic delay, and the extent of genetic lesions increase with chromosome size, thus explaining the correlation between interphase chromosome volume and radio-sensitivity. Lack of substantial radioprotecting effect of polyploidy in neoplastically transformed mammalian cells indicates residual capabilities to cease cell proliferation by mechanisms related to terminal differentiation, thus offering clues to tumour therapy.

Preleukemic proliferative changes in murine bone marrow after single and multiple 7,12-dimethylbenz(a)anthracene (DMBA)-applications.

The effect of a leukemia-inducing treatment on early changes in kinetic parameters of murine bone marrow cells were investigated. Mice were treated i.p. one, four and eight times at biweekly intervals with 1 mg DMBA. Up to nine weeks after the last injection, CFU-S number, proliferation ability of bone marrow cells (PF), cell doubling time (td) and the compartment ratio (CR) were measured. Following multiple DMBA injections, CFU-S number and PF were decreased whereas CR and td increased, thus indicating persisting stem cell injury and proliferative compensation in the hemopoietic amplification compartment. A single DMBA injection had no effect. It is concluded that a first DMBA injection induces cytotoxic (and genotoxic) damage in the bone marrow leading simultaneously to a strong proliferation stimulus and a hindered proliferation ability of HSC, some of which will be predisposed for further mutagenic treatment. The following DMBA injections meet strongly proliferating HSCs, thus enhancing the probability for the loss of proliferation control/terminal differentiation.

Magnetic field exposure of marrow donor mice can increase the number of spleen colonies (CFU-S 7d) in marrow recipient mice.

Transplantation of bone marrow cells of magnetic-field-exposed mice led to increased numbers of spleen colonies (CFU-S 7d) in conditioned recipient mice (Peterson et al. 1986). Here we report on the dependence of this phenomenon on body temperature, field strength and exposure time. It was found that the effect can only be seen when the body temperature is 27 degrees C, the field strength not less than 1.4 T and the exposure time at least 15 min. It is suggested that the magnetic field increases the number of spleen colonies either directly by affecting membrane components (receptors) responsible for the seeding of the transplanted stem cells to the recipient spleens or indirectly affecting radical/redox-systems that may have a regulatory function in the stem cells.

Terminal differentiation of human fibroblasts is induced by radiation.

In order to analyze the effect of various kinds of radiation on the terminal differentiation processes of fibroblasts in culture, both human skin and lung fibroblasts were irradiated with electromagnetic non ionizing as well as ionizing radiation in clonal and sparse mass culture systems. As analyzed by cell biological (cell type frequencies), biochemical (collagen synthesis) and molecular markers (expression of protein PIVa) human skin and lung fibroblasts are induced to differentiate prematurely into terminal postmitotic cells. Thus, both electromagnetic and ionizing radiation induce terminal differentiation in cultured cells. These data add some new aspects for the interpretation of radiation effects on cells, e.g., in clinical therapy, as well as for the development of normal tissue responses during early and late effects after radiotherapy.

Heritability estimates of pregnancy rate in beef cows under natural mating.

Observations of 3,029 matings over 17 yr on an Ozark upland range were used to estimate heritability of pregnancy rate in Angus, Hereford and Polled Hereford cows. Pregnancy rate, the percentage of cows exposed that produced a live calf in the spring, was transformed using the empirical logit transformation and then analyzed for each breed separately by weighted least squares using a mixed model procedure. A numerator relationship matrix for sires of cows was incorporated into the sire model to account for relationships among sires. Variation among years significantly affected pregnancy rate in all three breeds. Age of dam significantly affected pregnancy rate in the Angus and Hereford groups. Paternal half-sib estimates of heritability from the observed binary data (h2b) for pregnancy rate were calculated on first-calf heifers and mature cows for each breed. Respective h2b estimates for heifers and mature cows were .17 and .09 in the Angus group, .04 and .01 in the Hereford group and .05 and .05 in the Polled Hereford group. The heritability estimates when binary records were transformed to the probit scale (h2) were .04 +/- .003 and .02 +/- .001 for Angus, .01 +/- .002 and 0 for Hereford and .01 +/- .001 and .02 +/- .001 for Polled Hereford for heifers and mature cows, respectively. Heritability estimates in this study are in agreement with the literature, indicating little opportunity for improvement in pregnancy rate by selection within a breed.

Early and late effects in the bone marrow of mice following 2 Gy (6 MeV) neutron irradiation.

Following 5 Gy gamma irradiation, residual damage in bone marrow persisted up to one year and was ascribed to genetic defects in hemopoietic stem cells (von Wangenheim et al. 1986). To see whether high LET radiation is more efficient in inducing late effects, mice were whole-body irradiated with a single dose of 2 Gy neutrons (E = 6 MeV) and femoral cellularity, CFU-S number, proliferation ability of bone marrow cells (PF) and the compartment ratio (CR), i.e. the splenic 125-iodo-deoxyuridine incorporation per transfused CFU-S were measured up to one year after the radiation insult. Within 12 weeks, femoral cellularity, PF and CR recovered to control or near-control level, whereas CFU-S numbers remained significantly below control. No further recovery was observed. On the contrary, PF and CR deteriorated again after 12 and 26 weeks, respectively. CFU-S per femur tended to decrease as well. Thus it is demonstrated that a single dose of 2 Gy 6 MeV neutrons causes significant injury in function (PF) and structure (CFU-S numbers, CR) of bone marrow which persisted up to one year. While this residual injury can be attributed to genetic defects in hemopoietic stem cells, its increasing expression is probably due to late evolving damage in microenvironmental cells. The RBE of 6 MeV neutrons for the introduction of late effects in the bone marrow is in the range of 3.

Hemopoietic regeneration in murine spleen following transfusion of normal and irradiated marrow: different response of granulocyte/macrophage and erythroid precursors.

To investigate cell proliferation in regenerating spleen, bone marrow of normal and gamma-irradiated donor mice (3 weeks after 5 Gy) was transfused into lethally irradiated recipients. In the donors and in the recipient spleens numbers of CFU-S and progenitor cells were determined. In the irradiated donors the progenitors were at control level after 3 weeks of recovery although CFU-S were still at 50% of control. Recipients of the irradiated marrow received therefore an increased proportion of progenitors. CFU-C appeared to be self-renewing and/or increased in number due to enhanced CFU-S differentiation, but not the erythroid progenitors. CFU-S self-renewal was reduced after 5 Gy. The data suggest that cell differentiation and maturation proceed during early splenic regeneration. The quantity of CFU-C does not necessarily mirror the situation in the stem cell compartment.

5-Fluorouracil treatment after irradiation impairs recovery of bone marrow functions.

In mice, persisting radiation-induced growth retardation of hematopoietic tissue suggested that at least part of the surviving stem cells are genetically injured. Additional mitotic stress some time after the radiation insult might remove injured stem cells, thus improving the overall recovery of the irradiated bone marrow. Mice were treated with 5 Gy whole-body gamma irradiation. Two weeks later half of the animals were injected i.v. with 150 mg/kg 5-fluorouracil (5-FU), the other half remained untreated (5 Gy-controls). 2 or 10 weeks later, femoral cellularity and CFU-S content, proliferation ability of transplanted bone marrow and the compartment ratio (CR; ratio of splenic IUdR incorporation at day 3 and number of CFU-S transfused) were determined. Four weeks after 5 Gy and 2 weeks after 5-FU treatment all parameters showed significant impairment of recovery. 12 weeks after 5 Gy and 10 weeks after 5-FU CFU-S and CR were still reduced compared to the 5 Gy-controls. 5-FU treatment of unirradiated mice did not produce permanent effects on the quality of stem cells or the hematopoietic microenvironment. It is concluded, therefore, that an increased proliferation stimulus does not aid in the removal of injured CFU-S and may even impair recovery of bone marrow functions by increasing the proportion of genetically injured stem cells which continue proliferation.

Persisting radiation effect on murine 7-day and 12-day CFU-S.

If radiation-induced reduction in the proliferative ability of bone marrow cells is due to a change in the age structure of stem cells, it might be measurable by an increase in the ratio of 7- to 12-day spleen colonies. This increase has not been detected at either three weeks or one year after 5 Gy gamma-irradiation. It was found, however, that diameters of colonies and weights of hemopoietic tissue per colony were reduced after both periods of recovery. Thus, slow colony growth may have masked a change in the stem cell age structure. It is concluded that injury persists in a proportion of stem cells that continues the production of progeny and may contribute to late effects. Persisting cellular injury, in modified form, may affect all mitotically active tissues.

Residual radiation effect in the murine hematopoietic stem cell compartment.

Stem cells surviving radiation injury may carry defects which contribute to long-term effects. The ratio of 125-iododeoxyuridine (IUdR) uptake into spleens of lethally irradiated recipient mice between day 3 and day 5 after cell transfusion revealed reduced proliferative ability (PF) of spleen seeding cells in parallel with reduced CFU-S content of donors throughout the study period of one year after 5 Gy gamma irradiation. Additional data aided in evaluating possible mechanisms of PF reduction. Within the range of the graft sizes used, PF was independent of the numbers of cells or CFU-S transfused. Radiation-induced increase in loss of label between days 3 and 5 and prolonged doubling time of proliferating cells indicated enhancement of cell maturation and increase in mitotic cycle time. Increased IUdR uptake per transfused CFU-S suggested extra divisions of transit cells due to insufficiency in the stem cell compartment. It is concluded that persisting defects in surviving stem cells interfere in a complex way with cell proliferation in the hemopoietic system.

Long-term residual radiation effect in the murine hematopoietic stem cell compartment.

For the measurement of long-term residual radiation effect in the murine hematopoietic system a test system was developed that quantifies the proliferation ability of progeny of spleen repopulating cells by the proliferation factor (PF). The PF expresses the ratios of 125IUdR incorporation in the recipient spleens at days 3 and 5 following cell transfusion, thus measuring the relative increase in number of proliferating cells. Following 500 rad whole-body gamma irradiation, PF recovered up to 6 months and remained thereafter, on the average, at 80% of control. Recovery of the number of 7-day CFU-S was similar to recovery of PF. Various studies were aimed at elucidating the reasons for reduction in PF. Loss of incorporated 125IUdR activity from spleens between days 3 and 5 after cell transfusion indicates loss of mature labeled cells. When the doubling time of proliferating cells of CFU-S progeny (td) is corrected for cell loss, td for control bone marrow approaches mitotic cycle time in normal bone marrow as was found elsewhere. Following 500 rad, both cell loss and td were initially increased and recovered in parallel with PF and number of CFU-S. Reduction of PF could be brought about by radiation-induced increase in transient CFU-S with the consequence of increased loss of mature cells between days 3 and 5. This possibility was excluded by the observation that 1 year after 500 rad the number of colonies per spleen did not decrease from day 7 to day 12 after cell transfusion, as was expected from a higher proportion of transient CFU-S, but increased more than in the controls. Measurement of these 12-day colonies showed a significantly reduced size. Average progeny from irradiated CFU-S, apparently, grow more slowly. It is concluded that sublethal injury resides in stem cells, increases mitotic cycle time, and causes precocious loss of cells from spleens probably by enhanced differentiation and maturation due to interference with endocellular control of cell proliferation and differentiation. Probably the observed recovery proceeds via replacement of injured stem cells by less injured or normal stem cells.

Demand-control of proliferative activity in the hemopoietic system of lethally irradiated mice during transfusion-induced regeneration.

The extent of cell proliferation in the hemopoietic system after bone marrow transfusion of fatally irradiated mice depends on the regeneration of proliferative capacity. This may be modified by the demand for differentiated cells in the peripheral blood. This demand was suppressed by induction of transfusion plethora prior to 800 rad whole body irradiation and bone marrow transfusion. Controls were non-plethoric recipients. For 6 days the following parameters were measured: hemopoietic proliferation by the 125-iodo-deoxyuridine (125-IUdR) incorporation technique, CFU-S content and spleen colony histology. There are three general observations from spleen and marrow with respect to 125-IUdR uptake in plethoric mice: (1) initial higher 125-IUdR uptake, (2) reduced rate of increase of 125-IUdR incorporation, (3) this rate of increasing 125-IUdR uptake in spleen was more depressed than in marrow. On day 6 cellularity and CFU-S in spleen was below, and in marrow above that of the control. These data suggest that initially after fatal irradiation of control mice differentiation of transfused CFU-S predominates over proliferation. Later as the mice become anemic and erythropoietin is produced the stimulation to proliferate is greater in the control than in the plethoric mice in which erythrocytic proliferation is suppressed. These data suggest that there are multiple feedback loops that regulate regeneration in the spleen and the bone marrow. These differences may be connected with the microenvironment that preferentially initiates erythropoiesis in the spleen before the marrow and granulopoiesis in the marrow before the spleen.