Cytokine profiles in patients receiving wide-field + prostate boost radiotherapy (xRT) for adenocarcinoma of the prostate.

As a result of the association between ionizing irradiation and the induction of inflammatory and fibrogenic cytokines, circulating levels of IL-1alpha, macrophage colony stimulating factor (M-CSF) and TGFbeta were measured in a group of 37 patients who presented with well-defined adenocarcinoma of the prostate and were treated with wide-field pelvic (WFP) + prostate boost (PB) radiotherapy (xRT) according to RTOG protocols 94-08 and 94-13. First and foremost, patients with prostate cancer (PC) were found to have a significantly (p<0.05) elevated plasma level of the three cytokines prior to treatment. Moreover, during WFP + PB xRT, these circulating cytokine levels were further elevated, the elevation occurring in the form of cyclic waves; the concurrent waves of elevated IL-1alpha and M-CSF preceding that of TGFbeta. In addition to providing support for the existence of a humoral response to xRT in patients receiving WFP + PB xRT, the data demonstrated a significant correlation between the integral radiation dose (ID) and the temporal expression and magnitude of plasma IL-1alpha, M-CSF and TGFbeta levels in patients that had received 1-5 fractions (1.8-9Gy) of WFP + PB xRT. Thereafter, the appearance of elevated waves of cytokine expression in the patient's plasma continued independent of additional fractions of WFP + PB xRT.

Interleukin 1 alpha (IL-1) and macrophage colony-stimulating factor (M-CSF) accelerate recovery from multiple drug-induced myelosuppression.

Interleukin 1 alpha (IL-1) and macrophage colony-stimulating factor (M-CSF) interact synergistically to enhance the restoration of stem and progenitor subpopulations in murine marrow and, vis-a-vis, to accelerate hematopoietic recovery in 5FU myelosuppressed mice. Similarly, IL-1 is reported to accelerate recovery following myelosuppressive treatment with doxorubicin (AdR), cis-platinum (DDP) and cyclophosphamide (CTx). Studies were carried out in C57Bl/6 mice in order to determine whether IL-1 (+/- M-CSF) intervention was as effective against the myelosuppression experienced following 5FU-based multiple drug combinations. Maximal-tolerated doses (MTD) of AdR (10 mg/kg), DDP (8 mg/kg) or CTx (250 mg/kg) were administered either alone or in combination with 150 mg/kg 5FU. Cytokine intervention (q24 hours x 2) was initiated 24 hours later. Hematopoietic recovery was assessed by measuring the femoral content of the more primitive [IL-1 + IL-3 + M-CSF-responsive] HPP-CFC and the total granulocyte levels in the animals over a ten-day interval following treatment. MTDs of AdR, DDP and CTx, when compared with 5FU, produced only marginal levels of myelosuppression. As a result, cytokine intervention in animals treated with AdR, DDP or CTx resulted in only a modest, transient increase in the HPP-CFC and total granulocyte subpopulations when compared with their effect on 5FU--treated animals. Neither AdR, DDP nor CTx interacted with 5FU to significantly increase the cytotoxic effects of 5FU on the HPP-CFC or granulocyte subpopulations, and both IL-1 and IL-1 + M-CSF effectively stimulated hematopoietic recovery in all animals that received the 5FU--based drug combinations. However, the significant advantage (p < 0.05) achieved by combining IL-1 + M-CSF (vs. IL-1 alone) was only observed in animals that were treated with 5FU and either AdR or DDP. Furthermore, the initial stimulation of HPP-CFC recovery by IL-1 + M-CSF in animals that received DDP + 5FU, when compared with 5FU alone, was subsequently dampened. Although there were subtle, drug-related differences in the temporal response of the more primitive HPP-CFC and granulocyte populations to cytokine therapy, the data from this study demonstrated that abbreviated cytokine interaction can effectively accelerate hematopoietic recovery after combination drug therapy.

Secondary cytokines interact in sequence with interleukin-1alpha (IL-1alpha) with or without macrophage colony-stimulating factor (M-CSF) to further accelerate granulopoietic recovery in myelosuppressed animals.

Interleukin-1alpha (IL-1), by itself, accelerates both granulopoietic and thrombopoietic recovery in the 5-fluorouracil (5-FU) myelosuppressed mouse (FUM). As a primary cytokine, IL-1 also interacts in concert with macrophage colony-stimulating factor (M-CSF) to synergistically enhance hematopoietic recovery in the FUM. As part of our continuing interest in cytokine sequencing, studies were carried out to determine whether the addition of several secondary cytokines (GM-CSF, IL-3, and IL-6) to IL-1 (+/-M-CSF) would further enhance the stimulatory effects of the primary cytokine(s) on hematopoietic recovery in FUM. Throughout these studies, IL-1 (+/-M-CSF) was administered for 2 days to the FUM, and the secondary cytokines were given either in concert (days 1 and 2) or in sequence (days 3-6) or both with the primary cytokine(s). Based on the magnitude of 7-day post-5-FU granulocyte recovery, the results demonstrated that the synergistic effects of IL-1 + M-CSF treatment on granulopoietic recovery in FUM could not be duplicated by substituting either IL-3, IL-6, or GM-CSF for M-CSF. Nonetheless, the secondary cytokines were observed to enhance the stimulatory effects of IL-1 under the following administration schedules: (1) 2 days of IL-1, followed by a sequential treatment (days 3-6) with either IL-3 or IL-6, (2) 2 days of IL-1 + GM-CSF followed by an additional 4 days of GM-CSF alone, and (3) 2 days of IL-1 + GM-CSF followed by 3-4 days of a combination of GM-CSF and either IL-3 or IL-6. Although these cytokine treatment schedules led to an enhanced granulocyte recovery (vs. IL-1 alone) in FUM, the day 7 granulocyte numbers never exceeded those observed after 2 days of IL-1 + M-CSF. Similarly, granulocyte recovery in FUM receiving 2 days of IL-1 + M-CSF followed by either GM-CSF or IL-3 also was significantly greater than that observed with IL-1 + M-CSF alone. In contrast, however, the sequential administration of IL-6 with IL-1 + M-CSF, unlike IL-1, failed to further enhance granulopoietic recovery, suggesting that there may be an antagonism between IL-6 and M-CSF in the FUM. In summary, therefore, the secondary cytokines were found to interact more effectively when they were administered in sequence, rather than in concert, with both IL-1 and IL-1 + M-CSF.

Antioxidant enzyme activity in murine hematopoietic bone marrow following treatment with interleukin 1 alpha: influence of tumor.

To determine whether non-hematologic tumors influence the bone marrow's antioxidant enzyme response to the radioprotective cytokine interleukin 1 alpha (IL-1), studies were undertaken using BDF1 and Balb/c mice bearing small, medium or large Lewis lung carcinoma (LLCa) or EMT6 mammary carcinoma tumors, respectively. Results demonstrated that, similar to nontumor-bearing mice, treatment of tumor-bearing animals with IL-1 was associated with a significant increase in marrow MnSOD activity. However, the duration of this elevated activity was reduced as tumor burden increased, and this reduction may have an impact on IL-1's ability to radioprotect tumor bearing animals, especially when tumor burden is large. In addition to cytokine-mediated responses, significant tumor-related influences on the marrow's antioxidant enzyme status were seen. Notably, it was observed that the presence of tumor was correlated with a marked suppression of antioxidant enzyme activity. Surprisingly, however, the pattern of enzyme suppression was found to differ between the two tumor models studied both in temporal onset and in the number of enzymes involved. In conclusion, the data obtained from these studies on tumor-bearing animals demonstrate that there are both cytokine-related and tumor-related influences which can effect the antioxidant enzyme status of the hematopoietic marrow-influences which may have the potential to alter the marrow's ability to tolerate free radical-generating events, both endogenous (i.e inflammation, infection) and exogenous (i.e. radiation, certain chemotherapeutic drugs) in origin.

Enhanced platelet recovery in myelosuppressed mice treated with interleukin-1 and macrophage colony-stimulating factor: potential interactions with cytokines having megakaryocyte colony-stimulating activity.

Studies were carried out to determine whether the combination of IL-1 + M-CSF, similar to the effect of these cytokines on neutropenia, was able to reduce the duration of thrombocytopenia in the 5-fluorouracil (5-FU)-myelosuppressed mouse. In addition, comparisons were made between the in vivo effects of IL-1 + M-CSF and other "thrombopoietic" cytokines (e.g., IL-3, IL-6, and GM-CSF) that demonstrate some form of megakaryocytopoietic activity in vitro. Of the five cytokines studied, only IL-1 and IL-6, by themselves, were able to effect thrombopoietic recovery in the myelosuppressed mouse. IL-1, either when acting alone or interacting synergistically with M-CSF, was able to reduce significantly the period of thrombocytopenia, but the effects of IL-6 were restricted to enhancing platelet production during the period of rebound thrombocytopenia without altering the kinetics of thrombopoietic recovery. Moreover, none of the cytokine combinations studied were found to interact to reduce further the duration of thrombocytopenia beyond that observed with IL-1 + M-CSF. Nonetheless, IL-3, IL-6, and, to a lesser extent, GM-CSF were each able to interact with IL-1 + M-CSF to extend further the period of enhanced platelet production in the animal. However, scheduling studies suggested that these thrombopoietic cytokines interacted in sequence, rather than in concert, with IL-1 + M-CSF to enhance platelet production during thrombopoietic recovery. Furthermore, the data presented are consistent with the hypothesis that IL-1 + M-CSF initially acts on a multilineage, 5-FU-resistant target cell and that IL-6 (and possibly IL-3 and GM-CSF) serves as a secondary cytokine further to enhance platelet production during rebound thrombopoiesis in the 5-FU-treated mouse.

Temporal recovery of short-term repopulating HSC subpopulations in marrow following schedule-dependent administrations of IL-1 alpha and M-CSF.

Studies were carried out to establish the temporal effects of abbreviated administrations of IL-1 and IL-1 plus M-CSF as rescue agents on multipotential and short-term repopulating hematopoietic stem cell (HSC) subpopulations in murine marrow treated with a myelosuppressive dose of 150 mg/kg 5-FU. The recovery kinetics for high-proliferative-potential colony-forming cells (HPP-CFC), CFU-S8 and -S12, and both CFU-M and CFU-G compartments were monitored over a 14-day interval in 5-FU-treated bone marrow (FUBM) following daily cytokine injections over a 4-day interval. Both IL-1 and the coadministration of IL-1 and M-CSF rapidly enhanced the recovery of the HPP-CFC in FUBM to supranormal levels and maintained these levels for extended intervals. Moreover, since M-CSF was unable to influence the recovery of the HSC subpopulations in FUBM by itself, the results of the two cytokines amounted to a synergistic effect on the recovery of the HPP-CFC in FUBM and a reduction of severe neutropenia in the myelosuppressed animal. Scheduling studies demonstrated that these synergistic effects were restricted to those schedules in which M-CSF was coadministered with IL-1 during the first 2 days of cytokine rescue. Finally, the recovery curves generated for the HSC and CFU-M subpopulations in response to IL-1 (with or without M-CSF) also suggest that these cytokines may conceivably alter the normal balance between proliferation and differentiation within CFU-S8 and -S12 during the accelerated recovery of hematopoiesis in FUBM.

Stem cell responses in myelosuppressed mice following sequential treatment with recombinant human interleukin 1 (rHuIL-1), recombinant murine interleukin 3 (rMuIL-3) and recombinant human macrophage colony-stimulating factor (rHuM-CSF).

In vivo, recombinant human interleukin 1 alpha (rHuIL-1 alpha) + recombinant human macrophage colony-stimulating factor (rHuM-CSF) (IL-1 + M-CSF) effectively serves as a rescue agent for myelosuppression by enhancing the recovery of hematopoietic stem cell (HSC) subpopulations following treatment with 5-fluorouracil (5-FU). Because in vitro studies have suggested that hematopoietic recovery in 5-FU-treated bone marrow (FUBM) may proceed from a 5-FU resistant, (IL-1 + IL-3 + M-CSF-responsive) high proliferative potential HSC subpopulation of colony forming cells (HPP-CFC), studies were carried out to determine whether the addition of recombinant murine interleukin 3 (rMuIL-3) (IL-3) to either IL-1 or IL-1 + M-CSF would further enhance the recovery of HSC subpopulations in myelosuppressed C57Bl/6 mice. With the exception of the HPP-CFC, IL-3 dampened, rather than enhanced, the accelerated recovery of 8 d and 12 d colony forming units-spleen (8 d and 12 d CFU-S) and the committed macrophage progenitor (CFU-M) associated with in vivo treatment with IL-1 alone. Similarly, IL-3 interfered with the enhanced recovery of those HSC subpopulations in FUBM influenced by the synergistic interaction of IL-1 + M-CSF. This interference, however, was observed only when the rMuIL-3 was administered on day 2 or 3 of a four-day treatment with IL-1 + M-CSF. There was, however, no evidence that IL-3 exerted a negative influence on the restoration of granulocytes in the myelosuppressed animals. Moreover, sequencing studies provided data suggesting that the dampening effects of IL-3 on the synergistic interaction of IL-1 + M-CSF resulted from both an enhanced differentiation of the more primitive HSC subpopulations and a significant, but preferential, mobilization of the more mature 8 d CFU-S and CFU-M to extramedullary organs and that the mobilization of these more mature HSC subpopulations was temporally linked to their generation from the recovering HPP-CFC and 12 d CFU-S subpopulations.

Synergy between recombinant human IL-1 alpha (rHuIL-1) and M-CSF (rHuM-CSF) during the recovery of murine hematopoietic activity in myelosuppressed animals: abbreviated versus chronic administration of rHuM-CSF.

The ability of highly purified, recombinant human macrophage colony-stimulating factor (M-CSF) and recombinant human interleukin 1 alpha (IL-1) to rescue hematopoietic activity from the myelosuppressive effects of 5-fluorouracil (5-FU) was investigated in the C57Bl/6 mouse. IL-1 (q24 h x 4) stimulated granulopoietic recovery in the 5-FU-treated animals and reduced the period of severe neutropenia associated with this drug by 7 days. Chronic M-CSF administration (q24 h x 14), on the other hand, resulted in a modest retardation of granulocyte recovery, and, when combined with IL-1, the chronic administration of M-CSF significantly dampened the accelerated recovery of granulopoietic activity observed with IL-1 alone. Consistent with their effects on neutrophil recovery, IL-1 alone markedly enhanced the recovery of the granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU-GEMM), macrophage colony-forming units (CFU-M), and erythroid burst-forming units (BFUe) in the marrow, whereas M-CSF failed to demonstrate a significant influence on the restoration of these hematopoietic progenitors (with the exception of delaying the recovery of the BFUe). Unexpectedly, the combination of IL-1 plus M-CSF (q24 h, days 1-4) followed by M-CSF (q24 h, days 5-14) resulted in a more than additive stimulation of progenitor recovery in both the marrow and the spleen that was observed as early as day 3 following 5-FU treatment. Furthermore, in the absence of protracted M-CSF administration on days 5-14, the 4-day rescue with a combination of IL-1 plus M-CSF also resulted in a more than additive effect on the recovery from 5-FU-induced neutropenia. Collectively, these observations demonstrated that IL-1 and M-CSF can interact synergistically to stimulate granulopoietic recovery in the 5-FU-treated animal. However, the data also suggest that the continued administration of M-CSF following the 4-day IL-1 plus M-CSF rescue may interfere with the restoration of neutrophils in the myelosuppressed animal.

Absence of interleukin 1 alpha radioprotection in tumor-bearing animals: elevated plasma levels of prostaglandin E versus a preexisting primed marrow.

Recombinant human interleukin 1 (IL-1) administered as a "priming" agent 24 h prior to hematopoietically lethal doses of total body irradiation (TBI) confers radioprotection to normal C57B1/6 (B6) mice, but not to B6 tumor-bearing animals (TBAs) known to have altered hematopoietic steady states. Using the Lewis lung tumor (LLca) in the B6 mouse, studies were carried out to determine whether the failure of IL-1 to radioprotect the LLca TBA was related to a preexisting "primed" hematopoietic state in the TBA or resulted from inhibition of myelopoietic activity associated with the production of prostaglandin E (PGE) by, or in response to, the tumor. Both normal B6 and LLca B6 TBAs were injected (every 24 h x 1-5) with 100 micrograms of indomethacin (IND) prior to the administration of IL-1. A single treatment with IND was sufficient to reduce the elevated levels of PGE found in the plasma of the TBAs. After five treatments, IND reduced the PGE level to below that of controls. Neither the acute nor the protracted IND treatment, however, affected the expansion of the stem and progenitor cell compartments of the marrow in the LLca TBA. Furthermore, no evidence of restoration of the radioprotective properties of IL-1 was observed in TBAs pretreated with IND. Collectively, these data suggest that the failure of IL-1 to provide radioprotection to the LLca TBA is not a direct result of the elevated plasma PGE levels associated with growth of the LLca tumor. In addition, these studies provide insight into the importance of examining in vivo effects of biological molecules in altered, as well as normal, physiological states.

Marrow antioxidant enzyme activity in tumor-bearing and non-tumor-bearing mice following vincristine treatment.

Pretreatment or "priming" with vincristine (VcR) has been documented to radioprotect animals from whole body irradiation by accelerating recovery of hematopoietic marrow. The mechanisms underlying this phenomenon are unclear, but the marked similarities between priming with VcR and with immune stimulants such as endotoxin and glucan have led to speculation that VcR may be inducing such radioprotective immunoregulators as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The radioprotective ability of these cytokines, in turn, has been linked to an induction of the antioxidant enzyme manganese superoxide dismutase (Mn SOD). To establish whether priming with VcR is associated with induction of antioxidant enzymes, the activities of Mn SOD, copper-zinc (Cu-Zn) SOD, catalase (CAT), and glutathione peroxidase (GPX) were measured in the marrow of both LLca tumor-bearing and non-tumor-bearing mice given a priming dose of VcR. Results in non-tumor-bearing mice indicate that, similar to IL-1 and TNF administration, VcR treatment increases Mn-SOD activity, but not Cu-Zn SOD, CAT, or GPX activity. Furthermore, this increase occurs at the time VcR priming has been demonstrated previously to exhibit maximal radioprotection, suggesting that it may be contributing factor. However, VcR priming has been demonstrated to radioprotect both tumor-bearing and non-tumor-bearing animals, and no increase in Mn SOD activity (or the other enzymes monitored) was found in the tumor-bearing group. Rather, the presence of tumor significantly suppressed antioxidant enzyme activity. Collectively, the present data suggest that it is unlikely that increased antioxidant enzyme activity is directly involved in the VcR priming response.

Accelerated marrow recovery following total-body irradiation after treatment with vincristine, lithium or combined vincristine-lithium.

Accelerated post-irradiation recovery of hematopoietic marrow has been reported following treatment with lithium (Li) or vincristine (VcR). Because these two agents appear to exert their effects on different, albeit overlapping, hematopoietic populations, it was felt that combining them might lead to a wider spectrum of enhanced post-irradiation marrow regeneration. Results demonstrated that an accelerated recovery, which appeared to be additive in nature, was observed in the marrow following combined VcR-Li/4.5 Gy total-body irradiation. The combined schedule significantly enhanced post-irradiation recovery of white blood cells, 12-day spleen colony-forming units, erythroid burst-forming units, and fibroblastic colony-forming units over radiation alone; and recovery of marrow cellularity, multipotential colony-forming units (CFU-gemm) and granulocytic/monocytic colony-forming units (CFU-gm) over both radiation alone and either drug given singly with the 4.5 Gy. In addition, while data on the ability of regenerating stroma to support CFU-gm and CFU-gemm did not suggest that VcR was acting to enhance post-irradiation marrow recovery by increasing stromal production of hematopoietic growth factors, Li did appear to increase production of one or more of these factors, and this may be part of its mechanism of action.

Accelerated postirradiation recovery of hematopoietic marrow following priming with low doses of vincristine.

The present investigation is a continuation of efforts to characterize the radioprotective potential of priming with vincristine (VcR). In this study, the postirradiation recovery kinetics of the marrow's hematopoietic stem cell, progenitor cell, and stromal cell compartments were monitored following exposure to a range of sublethal radiation doses to determine (a) the optimal VcR/radiation intertreatment interval for achieving maximal hematopoietic protection, (b) whether this optimal interval is influenced by the dose of radiation administered, and (c) whether the radioprotection observed involves the hematopoietic stroma. The results demonstrate that the degree of radioprotection observed was significantly influenced by the scheduling of the VcR priming dose with respect to the radiation exposure. An intertreatment interval of 24 h provided maximal radioprotective benefit irrespective of the radiation dose administered. Additionally, the radioprotection following VcR priming appeared to be more the result of an accelerated recovery in the hematopoietic stem cell and progenitor cell compartments than a change in their intrinsic radiosensitivity. The data also suggest that this accelerated recovery was not a consequence of greater radioprotection of marrow stroma. Finally, the radioprotection observed following VcR priming did not appear to involve a selective lineage response by either the erythroid or the granulomonocytic progenitor compartments.

Altered radiosensitivity of hematopoietic stem cells by vincristine pretreatment: superoxide dismutase activity as a possible mechanism.

The effect of vincristine (VCR) on hematopoietic stem cell and progenitor compartments and its ability to induce transient periods of radioresistance was investigated so that we could ascertain the drug-radiation intertreatment interval affording optimal radioprotection and determine if its ability to induce increased levels of superoxide dismutase (SOD) is a potential mechanism for this radioprotection. Measurement of marrow stem cell and progenitor compartments demonstrated that these subsets displayed differential sensitivity to VCR and that this sensitivity appeared to be proportional to how "primitive" the subset was. Treatment with VCR prior to irradiation was observed to enhance significantly both 8- and 12-day spleen colony-forming unit recovery with maximal radioprotection occurring for a drug-radiation interval of 12-48 hours. Monitoring of copper-zinc SOD levels demonstrated an increase in activity following VCR that was localized in a fraction of the bone marrow enriched for stem cells and progenitors. The temporal pattern of this increase, however, did not correlate with the drug-radiation schedules affording optimal radioprotection, which indicates that other factors appear to be operative in this radioprotection as well.

Long-term consequences of chemotherapeutic agents on hematopoiesis: development of altered radiation tolerance.

The long-term effects of chemotherapeutic agents on subsequent radiation tolerance of the hematopoietic marrow were studied after a single injection of doxorubicin, 5-fluorouracil, or cyclophosphamide at a maximum tolerated dose. At designated intervals following drug treatment, drug-treated and age-matched control male B6D2F1 mice were exposed to 4.5 Gy of total-body irradiation, and the recovery kinetics of the stem cell (assayed at days 8 and 13 colony-forming spleen units) and progenitor (burst-forming erythroid units, and colony-forming erythroid and granulocyte/macrophage units) compartments were established. Response deficits were calculated for each compartment by comparison of treated and control recovery curves at 5 intervals over 32 weeks. Based on these response deficits, a number of conclusions were drawn: 1) There is selective drug specificity for the more primitive (13d) and mature (8d) CFUs subpopulations; 2) these sensitivities determine the temporal consequences of drug treatment on subsequent radiation tolerance in the marrow (e.g., acute, delayed, or long term); and 3) drugs that influence long-term radiation tolerance of the marrow are dose dependent and initially affect the more primitive stem cells. The data suggest that the initial lesion in the stem cell compartment, resulting in long-term enhancement of radiosensitivity, involves a major restriction (either in cell number or in genetic functionality) of the proliferative potential necessary for recovery from subsequent radiation insult.

Development of latent residual drug damage to the hematopoietic marrow during the subsequent growth of tumors.

Growth of the Lewis lung (LLca) tumor in BDF1 mice was found to be accompanied by a marked expansion of the multipotential stem (CFUs-8) and committed erythroid (BFUe) and myeloid (CFU-gm) progenitor cells of the marrow with a concomitant depression of more differentiated compartments. The long-term effects of adriamycin (AdR), busulfan (BU), cis-diaminedichloroplatinum II (DDP), and 5-fluorouracil (5-FU) on the LLca-induced expansion of the CFUs-8 and CFU-gm were investigated at eight weeks after drug treatment. Of the four drugs studied, only BU demonstrated a reduction of CFUs-8 at eight weeks after treatment and prior to tumor inoculation. However, all of the drugs were found to prevent the expansion of the CFUs-8 compartment after 16 days of tumor growth. BU also resulted in a depressed CFU-gm compartment at the time of tumor inoculation, while CFU-gm in ADR-, DDP-, and 5-FU-treated animals was either at control levels (AdR), or unexpectedly elevated (DDP and 5-FU). Similar to the observations made for CFUs-8, all drugs prevented the expansion of the CFU-gm associated with tumor growth. The data suggest that qualitative differences observed between the long-term effects of the drugs on the marrow compartments may be more accurately related to the temporal "fixation" of residual drug damage brought about by enhanced differentiation of a drug-limited pluripotential CFUs, than to the actual magnitude of hematopoietic damage.

Residual adriamycin (AdR)-induced hematopoietic damage: a consideration for subsequent radiotherapy.

The long-term effect of adriamycin (AdR) on the radiation response of hematopoietic marrow was studied at 16 weeks after treatment with a MTD (10 mg/kg) for the BDF1 mouse. The radiation response was monitored in both the "stem cell" (CFUs-8) and myeloid (CFU-gm, granulocyte, WBC) compartments, as well as the erythroid (BFUe, CFUe, HcT) compartments of the marrow for 14 days following a whole body dose (TBI) of 4.5 Gy X ray. At the time of irradiation, animal and spleen weight of AdR treated animals were reduced while HcT and WBC remained at control levels. At the same time the granulocyte and CFUs-8d compartments were depressed, while the BFUe compartment was expanded. The CFUe and CFU-gm compartments remained at control levels. For all marrow compartments, treatment with AdR 16 weeks prior to 4.5 Gy resulted in a radiation response deficit determined from the temporal recovery curves. The data suggest that manifestation of long-term AdR injury, at least through 16 weeks following treatment, is dependent on a subsequent stress of sufficient magnitude to enhance the proliferative activity associated with hematopoietic cell production and differentiation. A comparison is made between these observations and previously reported long-term drug-induced hematopoietic injury.

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow.

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFUc-GM compartment followed by an increase in the number of femoral CFUs. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFUe) compartment expanded to 168% of control, while the more differentiated (CFUe) compartment was reduced (45% of control at day 28). Reductions in both 59Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr X 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. The data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.

Effect of prolonged expansion of marrow progenitor compartments following doxorubicin (ADR) on subsequent radiation tolerance.

The long-term effects of a maximum tolerated dose of doxorubicin (ADR) (10 mg/kg, LD10/60) on the recovery of the hematopoietic compartments of the femoral marrow from radiation (450 rad) were investigated over a 32-week interval using a mouse model. Comparative radiation response curves, estimating hematopoietic proliferative potential, were used to establish response deficits (RD) for individual compartments of ADR-treated marrow. The RD data suggest that two potentially discrete lesions result from ADR treatment: one lesion associated with acute toxicity and a second developing 8-16 weeks after drug treatment. A depletion of the older hematopoietic stem cell (CFU-S8d) compartment was observed to accompany the development of the second lesion. Data are presented suggesting that both initial stem cell kill, as well as an accelerated aging of the CFU-S8d by prolonged expansion of progenitors, eventually lead to a depletion of stem cell reserve manifested by a progressive loss of radiotolerance with time.

Enhancement of colony-stimulating activity (CSA) on murine CFU-gm by the H-4-II-E2 (H4) tumor cell line of the rat.

The effect of the H-4-II-E2 (H4) rat tumor cell line on murine granulocyte/macrophage colony-forming units (CFU-gm) was studied in vitro using a bilayer (agar/methylcellulose) culture system over the tumor cell feeder and 10% colony-stimulating activity (CSA). The H4 cells demonstrated an amplification of CSA from several sources and of CFU-gm growth of murine marrow, including the CSA present in L-cell-conditioned medium (L-CSA; 200% of control). The amplification did not result from CSA produced by the H4 cell line, nor was cell-to-cell contact necessary for enhanced CFU-gm growth. Amplification of L-CSA was not mediated by endogenous or exogenous prostaglandin E concentrations in the in vitro system. Furthermore, incubation of the non-adherent marrow cell population with H4 tumor cells for 24 h prior to assaying for CFU-gm resulted in more colonies, independent of the continued presence of H4 tumor cells. The data suggest that the H4 tumor cells produce a readily diffusable, soluble factor that may amplify the effect of L-CSA on CFU-gm by stimulating a more primitive progenitor cell that expands the CFU-gm population.