SPARC modulates cell growth, attachment and migration of U87 glioma cells on brain extracellular matrix proteins.

We have identified secreted protein acidic and rich in cysteine (SPARC) as a potential glioma invasion-promoting gene. To determine whether SPARC alters the growth, attachment, or migration of gliomas, we have used U87T2 and doxycycline-regulatable SPARC-transfected clones to examine the effects of SPARC on (1) cell growth, (2) cell cycle progression, (3) cell attachment, and (4) cell migration, using growth curves, flow cytometry, attachment, and migration analyses on different brain ECMs, including collagen IV, laminin, fibronectin, vitronectin, hyaluronic acid, and tenascin. Our data indicate that SPARC delays tumor cell growth in the log phase of the growth curve. The clones secreted different levels of SPARC. The clone secreting the lowest level of SPARC was associated with a higher percentage of cells in G2M, whereas the clones secreting the higher levels of SPARC were associated with a greater percentage of cells in G0/G1. In comparison to the parental U87T2 clone, the SPARC-transfected clones demonstrated increased attachment to collagen, laminin, hyaluronic acid, and tenascin, but not to vitronectin or fibronectin. SPARC-transfected clones also demonstrated altered migration on the different extracellular matrix proteins. The modulation of migration, either positive or negative, was associated with changes in the level of secreted SPARC. These data suggest that SPARC may modulate glioma proliferation and invasion by modulating both the growth and migration of glioma cells.

Cellular uptake of a novel cytotoxic agent, cryptophycin-52, by human THP-1 leukemia cells and H-125 lung tumor cells.

Cryptophycin (CP) is a newly developed anticancer agent isolated from the terrestrial cyanobacteria of the genus Nostoc. CP is a mitotic inhibitor, causing cells to accumulate in mitosis with the disappearance of intracellular microtubules. In this report, we studied the interaction and uptake of a new synthetic CP analog, CP-52, with 2 human tumor cell lines, THP-1 and H-125. In vitro colony-forming assay showed that CP-52 has antiproliferative activity against THP-1 and H-125 cell lines with IC50 of 0.1 ng/ml and 20 microg/ml, respectively; i.e., THP-1 cells are 200,000 times more sensitive to CP-52 than H-125 cells. The uptake of CP-52 by the target cells was carried out using tritiated CP-52 (3H-CP-52). The uptake of 3H-CP-52 by both THP-1 and H-125 cells was rapid, reaching a maximum within 20 min. Dissociation experiments showed that CP-52 interacts with the target cells irreversibly, presumably by binding to specific cellular sites with high affinity. With increasing doses of 3H-CP-52, the uptake was found to be saturable, reaching a steady state as the concentrations of 3H-CP-52 were raised to about 20 microg/ml. Under this condition, the maximal values of CP-52 uptake by THP-1 and H-125 cells was estimated to be 27 and 136 ng/10(5) cells, respectively. The uptake and accumulation of 3H-CP-52 with the target cells was effectively inhibited by prior treatment with unlabeled CP-52 and, to a lesser extent, vinblastine and taxol but not adriamycin, colchicine or mitomycin. In addition, the binding of 3H-CP-52 to purified tubulin was inhibited by vinblastine but not taxol. This finding suggested that CP-52 and taxol interact and bind to distinct regions of tubulin molecules. Further, it suggests that, in addition to tubulin, other intracellular and/or membrane components are involved in mediating the binding of CP-52.

Influence of rhG-CSF scheduling on megakaryocytopoietic recovery following 5-fluorouracil-induced hematotoxicity in splenectomized B6D2F1 mice.

Recombinant human granulocyte colony-stimulating factor, rhG-CSF, is widely applied to ameliorate neutropenia following chemotherapy. However, rhG-CSF can exert negative effects on megakaryocytopoiesis that might cause a delay of megakaryocyte recovery. Therefore, the present study was designed to test different rhG-CSF administration protocols with regard to their megakaryocytic inhibitory potential in a 5-fluorouracil (5-FU)-induced experimental model system. Splenectomized B6D2F1 mice received a single injection of 5-FU (150 mg/kg) on day 0 followed by 50 micrograms/kg/day rhG-CSF given daily for either zero, four, or eight days. Five days after 5-FU, bone marrow and blood hematopoiesis were reduced significantly when compared with controls, independent of whether or not animals received rhG-CSF. However, nine days after 5-FU, granulopoietic recovery from 5-FU-induced toxicity was faster for rhG-CSF-treated versus untreated mice as demonstrated by higher values for colony forming unit-granulocyte macrophage (CFU-GM) and granulocytes (CFU-GM: 7.2 +/- 0.4 versus 5 +/- 0.6 x 10(4)/femur, granulocytes: 4.3 +/- 2 versus 1.4 +/- 0.4 x 10(5)/ml, respectively). Furthermore, significant mobilization of CFU-megakaryocyte (CFU-Meg) and CFU-GM into the peripheral blood was induced by the eight-day administration of rhG-CSF following 5-FU (day 9: 911 +/- 102 CFU-Meg/ml, 2330 +/- 152 CFU-GM/ml). However, megakaryocytic cells in these same mice were considerably lower when compared with those of animals receiving no rhG-CSF (CFU-Meg: 2.7 +/- 0.2 x 10(3) versus 4.2 +/- 0.2 x 10(3)/femur; small acetylcholinesterase positive (SAChE+) cells: 4.9 +/- 0.3 x 10(3) versus 7.3 +/- 0.9 x 10(3)/femur; megakaryocytes: 2.5 +/- 0.2 x 10(3) versus 4.1 +/- 0.7 x 10(3)/femur; platelets: 2.67 +/- 0.5 x 10(9) versus 3.1 +/- 0.5 x 10(9)/ml, respectively). On the other hand, the shortening of the rhG-CSF treatment from eight to four days caused a rapid granulopoietic recovery comparable to animals receiving eight days of G-CSF with no significant delay in megakaryocytic recovery when compared with mice treated with 5-FU alone; however, with four days of rhG-CSF, the mobilization of CFU into the peripheral blood was significantly less effective. Taken together, the results showed that a shortening of rhG-CSF treatment after chemotherapy is capable of ameliorating neutropenia without negatively affecting megakaryocytopoietic recovery. If, however, maximum recruitment of CFU into the peripheral blood circulation by rhG-CSF for subsequent harvest and transplantation is needed, any shortening of rhG-CSF administration is not advisable.

Flow cytometric analysis of DNA damage in nucleoids from cultured human breast epithelial cells treated with hydrogen peroxide.

Flow cytometric analysis of nucleoids is useful to investigate nuclear matrix-DNA associations in cells. We have now applied this technique to examine whether differences in nucleoid structure can be detected between tumor and normal-like human breast epithelial cells. We have previously shown that MCF-7 tumor and MCF-10A normal-like human breast cells exhibit different levels of endogenous oxidative DNA damage as well as differences in response to hydrogen peroxide. We therefore examined whether flow cytometric analysis of nucleoids can be used to detect both endogenous DNA damage and DNA damage induced by hydrogen peroxide in these cells. Nucleoids were prepared by lysis of MCF-7 and MCF-10A cells with a high-salt buffer. The size of the DNA loops around the nuclear matrix core was detected by measuring the forward light-scatter signal in the presence of ethidium bromide. The relaxation and supercoiling of DNA in the presence of low and high concentrations of ethidium bromide, respectively, was similar in MCF-7 tumor and MCF-10A normal-like cells. After treatment of cells with 100 microM and higher of hydrogen peroxide, there was a statistically significant increase in the forward light scatter from nucleoids prepared in the presence of high concentrations of ethidium bromide, and this increase was similar in both cell lines. The changes in the forward light scatter signal with increasing hydrogen peroxide concentration did not mimic the patterns of increases in single strand breaks or formation of 5-hydroxymethyl-2 '-deoxyuridine that we reported previously. This indicates that the flow cytometric technique probably detects other types of changes induced by hydrogen peroxide.

Quantitative measurements of the efficacy of new anti-cancer agents on fresh human AML cells by using multivariate flow analysis.

The testing of new human leukemia-specific drugs for activity against primary acute myeloid leukemia (AML) blasts is severely limited by the low and variable clonogenic potential of primary human leukemias in culture. To circumvent this problem, we have modified a previously described flow cytometric approach to permit the simultaneous determination of live/dead cells, and the quantitation of the surviving cell fraction as a ratio of viable cells in treatment and control groups. The method utilizes the combination of calceinAM as a probe for intracellular esterase activity (green fluorescence,) which has the advantage over carboxyFDA of pH insensitivity and superior signal-to-noise ratio, and propidium iodide (red fluorescence) as an indicator of plasma membrane integrity. Suspension cultures of AML blood and marrow samples from patients were treated with known active agents as well as several new agents arising from a clonogenic disk assay screen. Quantitative dose-response values obtained from surviving cell fractions assayed by flow cytometry at 24 h following drug exposure demonstrated the utility of this assay for quantitating drug-induced cytotoxic effects on primary human AML cells in short-term culture.

In situ radiation sensitivity of recombinant human granulocyte colony-stimulating factor-recruited murine circulating blood and bone marrow progenitors (colony-forming unit [CFU]-granulocyte-macrophage and CFU-megakaryocyte): evidence for possible biologic differences between mobilized blood and bone marrow.

Increasing evidence especially stemming from peripheral blood progenitor transplantation studies points to a possible biologic difference between mobilized blood and bone marrow progenitor cells. The objective of this study was to compare the in situ radiation sensitivity of recombinant human granulocyte colony-stimulating factor (rhG-CSF)-recruited circulating granulopoietic (blood colony-forming unit-granulocyte-macrophage [CFU-GM(blood)]) and megakaryocytopoietic (blood CFU-megakaryocyte [CFU-Meg(blood)]) progenitors, with the nonmobilized fraction remaining in the bone marrow (CFU-GM(femur) and CFU-Meg(femur)). Splenectomized male B6D2F1 mice received 50 micrograms/kg/d rhG-CSF daily for 8 days to induce high levels of circulating progenitors, followed by either total body X-irradiation (TBI) or X-irradiation of the chest (CI) with 62.5, 125, 250, or 500 cGy. Progenitor cells were assayed 24 hours after irradiation. Circulating CFU-GM and CFU-Meg in the blood were decreased in a dose-dependent fashion by both TBI and CI, with TBI causing greater damage than CI. Average D0 values for TBI were 53 cGy for CFU-GM(blood) and 40 cGy for CFU-Meg(blood) D0 values for CI were 90 cGy for CFU-GM(blood) and 140 cGy for CFU-Meg(blood). As seen for blood progenitor cells, TBI caused a dose-dependent decrease of both CFU-GM(femur) (D0, 136 cGy) and CFU-Meg(femur) (D0, 148 cGy). However, radiation-induced bone marrow progenitor cell kill was significantly lower when compared with blood progenitors. Despite the fact that circulating blood elements only received a fraction of the total dose administered as Cl, the extent of blood progenitor kill caused by Cl was higher than the effects of identical TBI doses on bone marrow CFU. The results of this study showed that rhG-CSF-recruited CFU-Meg(blood) and CFU-GM(blood) were considerably more radiosensitive than femoral progenitors, thereby providing novel evidence for a biologic difference between rhG-CSF-recruited peripheral blood progenitors and the nonrecruited bone marrow CFU.

Effects of rhG-CSF, 5-fluorouracil and extramedullary irradiation on murine megakaryocytopoiesis in vivo.

The aim of this study was to systematically characterize possible rhG-CSF effects on the murine megakaryocyte-platelet system (untreated and recovering from chemotherapy or extramedullary irradiation). In untreated, splenectomized male B6D2F1 mice, rhG-CSF treatment (50 micrograms/kg/d for up to 8 d) markedly decreased femoral megakaryocytopoiesis. CFU-Meg, small acetylcholinesterase-positive (SAChE) cells, and megakaryocytes were significantly reduced to 35-70%; platelets, however, were not affected. Peripheral CFU-Meg and CFU-GM increased up to 200-fold. Following a single injection of 5-FU (150 mg/kg) on day 0, rhG-CSF (50 micrograms/kg/d) on days 1-8 suppressed the megakaryocytopoietic recovery as indicated by significantly lower platelet numbers on day 9. Granulopoietic recovery was accelerated by rhG-CSF. When rhG-CSF treatment was started on day 5, no beneficial effect on granulopoietic recovery was observed, but again platelet levels were significantly lower on day 9, indicating that within the first 4 d of rhG-CSF application, recruitment or lineage competition was not a critical event. To test for the effects of extramedullary irradiation on circulating progenitors, mice pretreated with 50 micrograms/kg/d of rhG-CSF for 8 d received irradiation to the chest with 500 cGy resulting in a substantial kill of circulating CFU-Meg and CFU-GM of up to 99%. However, this striking decrease of blood progenitors did not significantly affect their total body contents. This study indicates that rhG-CSF treatment can impair bone marrow megakaryocytopoiesis, which might be an important consideration for those clinical situations that carry a high potential for treatment-induced thrombocytopenia.

Differential expression of mast cell growth factor receptor (c-kit) by peritoneal connective tissue-type mast cells and tissue culture-derived mast cells.

The peritoneal cavity of the mouse is a major source of connective tissue-type mast cells (CTMCs). Flow cytometric analysis using biotinylated recombinant murine mast cell growth factor (rMuMGF) showed that 1 to 3% of the cells in the peritoneal cavity exhibited MGF receptor (MGFR) (or c-kit). CTMCs were the only cell types expressing MGFR in the peritoneal cavity, and every one of them expressed MGFR. More than half the peritoneal CTMCs retained the potential to proliferate in the presence of recombinant murine interleukin 3 (rMuIL-3), rMuIL-4, and rMuMGF and gave rise to pure, alcian blue-positive "mast cells," which actively expressed c-kit transcripts and MGFR. Flow cytometric analysis and receptor assay carried out at 4 degrees C showed that the number of MGFRs on culture-derived mast cells (CDMCs) was one-third that of peritoneal CTMCs (6 x 10(4) vs. 1.8 x 10(5) MGFR/cell). At 37 degrees C, the total number of membrane MGFRs detected in CDMC was two to three times more than that detected at 4 degrees C, indicating that nearly 70% of total MGFR in CDMCs, compared with only 40% in peritoneal CTMCs, existed as "cryptic sites" unable to interact with exogenous ligand at 4 degrees C. Thus, diminished expression of MGFR is one of the phenotypic characteristics associated with CDMCs.

Acute toxic effects of 3'-azido-3'-deoxythymidine (AZT) on normal and regenerating murine hematopoiesis.

The immediate hematopoietic response to 3'-azido-3'-deoxythymidine (AZT) was characterized in an unperturbed and regenerating mouse marrow model to identify the in vivo hematopoietic targets of AZT and test whether AZT toxicity is dependent on the proliferative activity of the hematopoietic targets. B6D2F1 mice received intravenous (IV) bolus injections of 30, 60, 120, and 240 mg/kg AZT. None of the doses induced consistent changes in the number of hematopoietic stem and progenitor cells. However, identical cumulative doses administered as an intravenous 24-hour infusion led to marked changes. Spleen colony-forming units (CFU-S) per femur were diminished to about 60%. Burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) were substantially reduced to about 15 to 35% at the two highest doses, whereas the femoral content of colony-forming units-granulocyte/macrophage (CFU-GM) and colony-forming units-megakaryocyte (CFU-Meg) was unchanged. When administered to mice whose marrow was regenerating from total-body irradiation (TBI) and subsequent bone marrow transplantation (high proliferative fraction), 240 mg/kg AZT caused considerable reductions of all hematopoietic cell stages even when given as a single IV injection. The results indicate that (1) the mode of application is critical for AZT hematotoxicity; (2) erythropoietic progenitors are the most sensitive to AZT toxicity; and (3) hematotoxicity increases with increasing proliferative activity.

The role of lymphoid cells in hematopoietic regulation.

The present review has summarized evidence supporting the existence of different lymphoid subsets with opposing effects on hematopoietic cell growth (Table 1); specifically; a) growth stimulation resulting from the release of interleukins and granulocyte-macrophage colony-stimulating factor (GM-CSF) by particular subsets of lymphocytes and resting natural killer (NK) cells and b) growth inhibition resulting from the release of interferon (IFN) and tumor necrosis factor (TNF) by stimulated NK, B and lymphokine-activated killer (LAK) cells and inhibitory T lymphoid subsets. The systematic examination of the physiologic relevance of lymphoid subsets would add an important element to a more comprehensive model of hematopoietic regulation that might hold promise in future clinical applications.

Identification of the c-kit ligand: end of the road for understanding aplastic anemia in steel mutant mice?

We here report the initiation of hematopoietic recovery in congenitally hypoplastic S1/S1d mice by the cytotoxic ablation of cells bearing the natural killer (NK) phenotype (NK 1.1+). The most striking finding was the early several-fold increase in the cycling fraction of stem and progenitor cells (with the exception of progenitors committed to megakaryocytopoiesis) in the anti-NK 1.1+ antibody-treated group. This increase resulted in an early, complete restoration of total marrow cellularity to the normal (+/+) littermate level. Our data suggest that NK 1.1+ cells exert functions critical to the negative control of hematopoietic cell proliferation in S1/S1d mice.

Sequential dacarbazine/cisplatin and interleukin-2 in metastatic melanoma: immunological effects of therapy.

Thirteen previously untreated patients with metastatic melanoma entered into a phase II chemo-immunotherapy trial were monitored immunologically during treatment. Treatment consisted of dacarbazine (DTIC) 750 mg/m2 and cisplatin 100 mg/m2 on day 1 followed by interleukin-2 (IL-2) 4 x 10(6) U/m2 by daily intravenous bolus on days 12-16 and 19-23. Cycles were repeated every 28 days. On days 1 (pretreatment), 12, 16, and 23 of each cycle, lymphokine-activated killer (LAK) cell and natural killer cell activity as well as total lymphocyte count and CD3, CD4, CD8, and CD56 lymphocyte subsets were analyzed. Despite pretreatment with full-dose cytotoxic chemotherapy, all patients were able to respond immunologically to IL-2. Spontaneous LAK cell activity was generated by the end of each course of IL-2 administration and persisted for at least 5 days thereafter. Lymphocytosis was maximum at 5 days after IL-2 administration and included increased numbers of all measured lymphocyte subsets. IL-2 administration caused a relative increase in CD56+ cells and a relative decrease in CD3+ cells. There was a direct correlation between the increase in LAK cell activity and the increase in CD56+ lymphocytes. Antitumor responses occurred in five patients but these responses did not correlate with any of the measured changes in LAK activity or lymphocyte subsets. DTIC and cisplatin administered in this schedule does not abrogate the immunological effects of IL-2.

Differential effect of L3T4+ cells on recovery from total-body irradiation.

We have examined the importance of L3T4+ (murine equivalent to CD4+) cells for hematopoietic regulation in vivo in unperturbed mice and mice recovering from total-body irradiation (TBI) using a cytotoxic monoclonal antibody (MoAb) raised with the GK 1.5 hybridoma. Ablating L3T4+ cells in "normal" (unperturbed) B6D2F1 mice substantially decreased the S-phase fraction (determined by in vivo hydroxyurea suicide) of erythroid progenitor cells (erythroid colony-forming units, CFU-E) as compared to the pretreatment level (10% +/- 14.1% [day 3 following depletion] vs 79.8% +/- 15.9%, respectively) with a corresponding decrease in the marrow content of CFU-E at this time to approximately 1% of the pretreatment value. Although the S-phase fraction of CFU-GM was decreased to 2.2% +/- 3.1% 3 days after L3T4+ cell ablation from the 21.3% +/- 8.3% pretreatment value, CFU-GM cellularity showed little change over the 3 days following anti-L3T4 treatment. Anti-L3T4 MoAb treatment had little or no effect on either the S-phase fraction or the marrow content of hematopoietic "stem cells" (spleen colony-forming units, CFU-S) committed to myeloerythroid differentiation. Ablating L3T4+ cells prior to a single dose of 2 Gy TBI resulted in significantly reduced marrow contents of CFU-S on day 3 and granulocyte-macrophage colony-forming units (CFU-GM) on day 6 following TBI, with little or no effect on the corresponding recovery of CFU-E. The present findings provide the first in vivo evidence that L3T4+ cells are involved in: 1) maintaining the proliferative activity of CFU-E and CFU-GM in unperturbed mice and 2) supporting the restoration of CFU-S and CFU-GM following TBI-induced myelosuppression.

Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry.

Different ploidy classes of rat megakaryocytes were sorted by flow cytometry from highly purified perfusion-fixed megakaryocyte cell suspensions prepared by sequential centrifugal elutriation and Percoll gradient centrifugation. Sorted cell populations were studied for the localization of platelet factor 4 (PF-4) probed with the monoclonal antibody 2E7 in order to clarify the relevance of PF-4 localization to the cytoplasmic and nuclear development of megakaryocytes. The relative numbers of labeled alpha granules and labeled alpha granule-related small vesicular structures (AGR-SVS) were quantitated using the gold-labeled antibody detection method and correlated with DNA content and cytoplasmic maturation in individual megakaryocytes. We determined that the stage of cytoplasmic maturation exerted a significant effect on the proportion of labeled alpha granules and labeled AGR-SVS. A significant interaction effect of stage and ploidy class resulted in the stage effect on proportion of labeled alpha granules being significant only in two of the three ploidy classes. The least mature cells present within each ploidy group exhibited PF-4 labeling mostly in SVS that were not related to alpha granules. During subsequent cytoplasmic maturation, more of the labeled SVS were seen related to alpha granules, with more of the mature alpha granules themselves becoming labeled. Polyploidization also affected the proportion of labeled AGR-SVS. Our data suggest that SVS play a role in the intramegakaryocytic transport of PF-4 into alpha granules. These data provide evidence of the complexity of megakaryocytic differentiation involving both cytoplasmic maturation and nuclear endoreduplication as reflected in PF-4 expression.

Stem cell recovery from cyclophosphamide-induced myelosuppression requires the presence of CD4+ cells.

Recently, we have reviewed studies regarding the growth-stimulating effect of CD4+ cells on haematopoietic cells in culture (Pantel & Nakeff, 1989a). In the present study we have tested the physiologic relevance of this interaction using a drug-perturbed mouse model. The long-term application of cyclophosphamide (CY, 30 mg/kg/d, five i.p. injections per week over 7 weeks) in B6D2F1 mice resulted in initial CY-induced cytotoxicity to CFU-S followed by the reestablishment to pretreatment values of the femoral content of CFU-S within 2-3 weeks of CY-treatment. An examination of CY-metabolism in these treated mice excluded a pharmacological explanation for the compensation of CY-cytotoxicity. However, a three-fold increase in the cycling fraction of CFU-S (determined by in vivo hydroxyurea suicide) was observed concomitant with a two-fold increase in the femoral content of L3T4+ cells (the murine equivalent to human CD4+ cells), as compared to the corresponding values in untreated mice. Ablating these L3T4+ cells in vivo by means of a cytotoxic monoclonal antibody (MoAb) to the L3T4 determinant resulted in a decrease in the cycling fraction of CFU-S from 56 +/- 8% to essentially zero and a decrease in the femoral content of CFU-S when comparing mice receiving either CY alone or CY plus MoAb, respectively. It would appear that the CY-induced increase in the proliferative activity of CFU-S requires the presence of L3T4+ cells. These data constitute the first in situ evidence for the physiologic relevance of immunocompetent L3T4+ cells as regulators involved in the recovery of stem cells from drug-induced myelosuppression.

Inhibition of hematopoietic recovery from radiation-induced myelosuppression by natural killer cells.

We have examined the role of natural killer (NK) cells in situ in the recovery of marrow hematopoiesis in B6D2F1 mice receiving various doses of total-body irradiation (TBI) as a well-characterized model for treatment-induced myelosuppression. Applying an in situ cytotoxic approach for ablating NK 1.1 cells, we have demonstrated that NK 1.1 cells differentially inhibit the recovery of hematopoietic stem cells (CFU-S) and their progenitor cells committed to granulocyte-macrophage differentiation from a sublethal dose of TBI (9 Gy) while not affecting the recovery of progenitor cells committed to either erythroid or megakaryocyte differentiation from TBI. However, recoveries of CFU-S and progenitor cells were unaffected by the ablation of NK cells prior to a moderate dose of TBI (2 Gy). These findings provide in situ evidence that NK cells are potential inhibitors of hematopoietic recovery from treatment-induced myelosuppression.

Differential effect of natural killer cells on modulating CFU-Meg and BFU-E proliferation in situ.

As a result of the striking discrepancy between the substantial amount of information on the role of natural killer (NK) cells derived from in vitro experimentation and the corresponding lack of data demonstrating their physiologic relevance, we have examined the importance of NK cells for the steady state production of hematopoietic stem and progenitor cells in situ. B6D2F1 mice received two 0.2-ml injections of ascites containing anti-NK 1.1 monoclonal antibody (anti-NK) directed to murine NK cells. Another group was treated similarly but received "control" ascites (CA) that was induced solely by injection of a mouse myeloma cell similar to the fusion partner of the NK 1.1 hybridoma. Two days after the last injection, we determined the number and cycling fraction (i.e., percentage of cells in S-phase determined by in vivo hydroxyurea suicide) of femoral stem cells (spleen colony-forming units; CFU-S) and committed granulocyte-macrophage (granulocyte-macrophage colony-forming units; CFU-GM), megakaryocytic (megakaryocyte colony-forming units; CFU-Meg), and erythroid (erythroid burst-forming units; BFU-E and erythroid colony-forming units; CFU-E) progenitor cells. The striking finding was the almost complete abolishment of the proliferation of CFU-Meg in the anti-NK group, resulting in a statistically significant (p less than 0.02) decrease in number to 37% of the CA control. In contrast, the cycling fraction of BFU-E was significantly (p less than 0.05) increased to 205% of the CA control with no increase in number. The number and cycling fraction of CFU-S, CFU-GM, and CFU-E in the anti-NK group were not significantly different from values in the control group. These findings add a novel aspect to the understanding of hematopoietic regulation by providing the first evidence for a differential effect of NK cells on the steady-state proliferation of CFU-Meg and BFU-E in situ.

Cyclophosphamide-induced enhancement of stem cell recovery from whole-body irradiation is radiation dose-dependent.

Although the enhancement of CFU-S recovery by CY pretreatment has been described for combinations of TBI greater than 4 Gy, no information exists as to whether such enhancement is operative at lower TBI doses (less than 3 Gy). B6D2F1 mice received intraperitoneal injections of CY (2 and 5 mg/mouse) at various time intervals prior to 2, 5 or 9 Gy TBI. The control group received the same dose of TBI without CY pretreatment. Six days after TBI, we determined the femoral content of CFU-S. Administration of 5 mg CY per mouse 3 days prior to 5 or 9 Gy TBI enhanced recovery of CFU-S with a substantial increase in CFU-S of 3- and 30-fold over control, respectively. In contrast, the administration of CY prior to 2 Gy TBI delayed recovery with CFU-S remaining at only 10 to 40% of control. This prolongation in CFU-S recovery was similar for CY doses of either 2 or 5 mg per mouse at all the time intervals tested (2, 3, 4 and 7 days prior to TBI). Our results demonstrate an enhancement of CFU-S recovery by pretreatment with CY for high doses of TBI that is reversed if the subsequent TBI dose is too low.

Ultrastructural localization of platelet factor 4 in rat megakaryocytes and platelets by gold-labeled antibody detection.

The pattern of distribution of platelet factor 4 (PF-4) was studied in rat platelets and megakaryocytes following immunohistochemical labeling with a monoclonal antibody (2E7) specific for PF-4 and visualization by a protein A-gold complex. We observed a heterogeneity in PF-4 expression among alpha granules with a minority of them being unlabeled by immunoelectron microscopy, a pattern similar in both mature, circulating platelets and developing megakaryocytes. Furthermore, the majority of the labeled alpha granules in both cell types displayed a unique, eccentric localization of the PF-4 that was mainly over the nucleoid region within each granule. This localization is similar to the microtubular localization in alpha granules reported previously for von Willebrand factor and yet distinct from the reported random distribution of fibrinogen. We also observed significant labeling of small vesicular structures in developing megakaryocytes that may be involved in the transport of PF-4 and its packaging in platelet alpha granules. This new information is important in relating patterns of PF-4 biogenesis in megakaryocytes to conditions of alpha granular dysfunction in platelets.

Lymphoid cell regulation of hematopoiesis.

It is clear from extensive in vitro data that different subsets of lymphocytes stimulate and inhibit the growth of hematopoietic stem and progenitor cells. In order to clarify the complexity of the network between regulatory lymphocytes and hematopoietic target cells, we have examined the stimulatory and inhibitory effects derived from different lymphoid subsets. The regulatory influence of lymphocytes is transmitted mainly through the release of cytokines including the interleukins, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-beta and the interferons, all of which have non-specific effects on a variety of hematopoietic cells. Since these cytokines amplify the effects of other, more lineage-specific cytokines (e.g., erythropoietin, thrombopoietin and granulocyte or macrophage colony-stimulating factor) on the proliferation and differentiation of progenitor cells, the present review supports the conclusion that lymphoid subsets play a critical role in ensuring an optimal hematopoietic response to specific demands.