Transmission electron microscopy of fetal rat brain cells during neoplastic transformation in cell culture.

Fetal rat brain cells were investigated by transmission electron microscopy during neoplastic transformation in long-term cell culture. Before transfer of the cells to culture, BD IX rat fetuses were treated with a single transplacental pulse of N-ethyl-N-nitrosourea (75 micrograms/g body wt) on the 18th day of gestation. During the early stages (3--4 mo), both glia-like and neuron-like cells were present in the culture, and after 2 months they formed complex aggregates ("nodules"). In contrast, corresponding secondary control cultures consisted of flat, epithelioid neural cells without neuron or astrocyte differentiation. After 3 months, cells with neuron morphology gradually disappeared. Some of the remaining cells contained many autophagosomes. After 5 months, rapid proliferation of rather homogeneous, glia-like populations was accompanied by reduction of microfilament bundles and microtubules, as well as atypical nuclei. Ability to form tumors upon sc implantation into syngeneic hosts was not observed until about 3 months later.

Neoplastic transformation of fetal rat brain cells in culture after exposure to ethylnitrosourea in vivo.

A single, transplacental pulse of N-ethyl-N-nitrosourea (ENU; 75 mug/g body wt) to the fetal (18th day of gestation) BD IX rat led to death with malignant tumors of the central and peripheral nervous system after a median time of approximately 195 days. In contrast to untreated control cells, dissociated brain cells transferred to long-term cell culture 20-90 hours after the ENU pulse became tumorigenic after approximately 200 days, as assayed by reimplantation into baby BD IX rats. This was preceded by a characteristic sequence of phenotypic alterations (termed "stages I-IV"). During early primary culture (stage I), both ENU and control cultures exhibited stationary glia-like cells on a growing layer of epithelioid (possibly glia precursors) and few fibroblast-like cells. Stage II (approximately 10th-40th day) was characterized by a constant proportion of glia-like cells in the ENU cultures and by their disappearance in the controls. During stage III (approximately 40th-100th day), slowly proliferating glia-like cells in the ENU cultures formed "piled-up" foci. They could then be removed from the underlying cell layer and cultured separately. Transition to stage IV (approximately 100th-200th day) was marked by proliferation of morphologically altered cells, which subsequently acquired the capacity of form colonies in semisolid agar and finally became tumorigenic (stage V). This system may represent a model for the study of malignant transformation.

Surface structure of fetal rat brain cells during neoplastic transformation in cell culture.

The surface microstructure of fetal rat brain cells undergoing neoplastic transformation in long-term cell culture after a single transplacental pulse of 75 microgram N-ethyl-N-nitrosourea/g body weight to the fetal (18th day of gestation) BD IX rat was investigated by scanning electron microscopy. After about 3 weeks of culture, N-ethyl-N-nitrosourea-pretreated fetal rat brain cells showed focal proliferation of neural cells on an underlayer of flat, epithelioid cells. The neural cells exhibited varying forms of numerous dorsal ruffles and an increased number of other surface microprojections. Between the 40th and the 100th day, nodules of bipolar and multipolar neural cells were observed with a complex surface microstructure including many blebs and ruffles and an increased number of microvilli. After 100-210 days, more rapidly proliferating, morphologically altered cells formed "piled-up" foci, which resulted in a homogeneous population of cells with numerous long microvilli, large ruffles, and blebs over the whole surface. The cells retained the same altered surface structure until tumorigenicity after reimplantation into the syngeneic host was first observed (approximately 273 days). Surface alterations characteristic of the neoplastic cells were thus observable more than 100 days before the cells became tumorigenic.

Phagocytic capacity of normal and malignant rat glial cells in culture.

The phagocytic capacity of 4 continuous rat glioma cell lines (BT2C, BT4Cn, BT5c, and 9L) and normal BD IX fetal rat glial cells in culture has been studied. This was done by flow cytometric measurements of single cells from monolayer cultures having ingested fluorescent bacteria, zymosan particles, red blood cells, or fragments of normal glial cells. In addition, phagocytosis was studied in a three-dimensional culture system. The BT4Cn, BT5C, and 9L cell lines were tumorigenic and invasive both in vivo and in organ culture in vitro. In contrast, BT2C has shown variable tumorigenicity and does not seem to be invasive. The phagocytic capacity of the cell lines was compared to their destructive properties during invasion. Depending on the particle type, 30-40% of the normal glial cells were phagocytic. The fractions of phagocytic glioma cells were dependent on the particle type and the prey load. Of the invasive cell lines, BT5C showed high phagocytic activity both in monolayer and three-dimensional cultures. Two of the invasive cell lines (BT5C and 9L) had about the same fraction of phagocytic cells as normal glial cells. These 2 cell lines showed highly destructive growth during invasion. In contrast, the third invasive cell line (BT4Cn) had almost no phagocytic cells. The BT4Cn cells showed single-cell invasion with little destruction of target tissue. The noninvasive cell line (BT2C) showed low phagocytic activity, and almost no destruction was observed in the border zone between tumor cells and normal tissue. Phagocytosis seems to be an inherent property of both normal and malignant glial cells, although the fraction of phagocytic cells varies from one cell line to another. In organ culture high phagocytic capacity of invasive glioma cells seems to be related to destructive activity on the normal brain tissue during invasion.

Invasion of rat neurogenic cell lines in embryonic chick heart fragments in vitro.

Invasive properties of 15 continuous neurogenic rat cell lines were investigated in vitro and were compared to their tumorigenicity in inbred BD IX rats. The cell lines were obtained by treating animals with a single transplacental dose of the carcinogen N-ethyl-N-nitrosourea (ENU) and 1) subsequently transferring brain cells into cell culture shortly after treatment or 2) explanting the resultant tumors from the offspring to monolayer cultures. In addition, one continuous nonneoplastic rat fibroblast line and three samples of untreated fetal rat brain cells were investigated. Cells from monolayer cultures were suspended and allowed to form aggregates for 24 hours on a gyratory shaker. The cell aggregates were then brought into contact with and allowed to attach to fragments from 9-days embryonic chick heart and were cultured on a gyratory shaker. All tumorigenic cell lines invaded the heart fragments, as characterized by progressive replacement of heart cells by invading cells. The heart tissue degenerated irreversibly. Nontumorigenic cell lines did not show invasiveness in vitro. Confrontation of cell aggregates and heart fragments in organotypic culture appeared to be a useful method to study directly the invasive properties of malignant transformants of neurogenic cells. The method might also permit prediction of tumorigenicity in the animal.

Interaction between rat glioma cells and normal rat brain tissue in organ culture.

A system for coculture between normal rat brain fragments and multicellular spheroids of rat glioma cells is described. The tumor cells were derived from fetal BD IX rats treated transplacentally with the carcinogen N-ethyl-N-nitrosourea (CAS: 759-73-9). Brain fragments were obtained from fetal BD IX rats and precultured for 20 days before confrontation with multicellular tumor spheroids. The cocultures were grown in nonadherent stationary organ culture for 30 days. Due to morphologic similarities between normal brain cells and tumor cells, the tumor cells were labeled with tritiated thymidine, which made them easily recognizable in autoradiographs. The two structures adhered to each other, and glioma cells progressively invaded and replaced the normal brain tissue. Invasion and replacement are characteristic features of brain tumors in vivo. Therefore, this organotypic and syngeneic model may be useful for investigation of these phenomena outside the body.

Glutathione content in human bone marrow and circadian stage relation to DNA synthesis.

DNA synthesis and contents of reduced glutathione (GSH) and oxidized glutathione were determined every 4 hours during a 24-hour period in 70 human bone marrow samples from 10 healthy males. The mean GSH contents during the sampling periods were low, varying from 1.94 to 3.27 nmol/mg protein between the subjects; the mean values for all samples were 2.54 +/- 0.06 nmol/mg protein. The GSH content varied markedly within the individual according to circadian stage (31.0% to 90.2%; mean, 51.4%). Between individuals the mean percentage of cells in DNA synthesis varied from 10.6% to 14.5%, but there was an intraindividual circadian stage-dependent variation, ranging from 48.9% to 274.0% (mean, 126.6%), relative to the lowest value. After adjustment for a slight phase difference between GSH content and DNA synthesis observed for some of the subjects, a statistically significant correlation was found between the GSH content and the fraction of cells in DNA synthesis. The myelosuppressive effect of many chemotherapeutic agents assumed to be detoxified by GSH-dependent mechanism(s) should be considered in the light of the low GSH content in human bone marrow, the circadian variation of DNA synthesis, and the circadian stage-dependent relationship of the GSH content and DNA synthesis.

Leptomeningeal tissue: a barrier against brain tumor cell invasion.

BACKGROUND: Primary brain tumors are characterized by an extensive infiltrative growth into the surrounding brain tissue. This process is confined to the central nervous system, and tumor cell metastasis to other organs is rare. However, other tumors of non-neural origin may frequently metastasize to the central nervous system. PURPOSE: The purpose of the present study was to examine the invasive behavior of different glioma cells into tissues of neural (brain aggregates) as well as non-neural origin (leptomeningeal tissue). Using the same target tissues, the invasive characteristics of two neural metastatic tumors (one malignant melanoma and one small-cell lung carcinoma) were also studied. This direct comparison of the invasive behavior between tumors of neural and non-neural origin provides valuable information regarding the mechanisms of glioma cell dissemination in the central nervous system. METHODS: The in vitro invasive behavior of human tumors of the central nervous system into human leptomeningeal tissue as well as into normal rat brain tissue was studied. For this purpose, a co-culture system consisting of tumor biopsy specimens, human leptomeningeal cell aggregates, and brain cell aggregates was established. Three glioblastomas, one oligodendroglioma, one meningioma, one small-cell lung carcinoma, and one malignant melanoma were studied. RESULTS: In co-cultures of gliomas and leptomeningeal cell aggregates, a well-defined border between the two tissues was observed. The brain cell aggregates, in contrast, were consistently invaded by the glioma cells. The brain metastases showed a different invasion pattern. The metastatic cells invaded and progressively destroyed leptomeningeal cell aggregates, whereas they did not invade the brain cell aggregates. Upon confrontation of the leptomeningeal tissue with the meningioma, a fusion of the two tissues was observed. Immunostaining of the leptomeningeal tissue showed a strong expression of the basement membrane components fibronectin, collagen type IV, and laminin with no expression of glial fibrillary acidic protein, neuron-specific enolase, or S-100 protein. CONCLUSIONS: The present study indicates that there may be important biologic differences between the invasive behavior of gliomas and non-neuroepithelial tumors. Our co-culture experiments suggest that leptomeningeal cells and associated acellular components may constitute a barrier against glioma cell invasion. However, this barrier may not be functional for metastatic tumors to the brain. The presence of glioma cells within the leptomeninges should not necessarily be taken as evidence of aggressive growth or as an indicator of malignancy.

Identification of a regulatory peptide distinct from normal granulocyte-derived hemoregulatory peptide produced by human promyelocytic HL-60 leukemia cells after differentiation induction with retinoic acid.

It has been found that leukemia cells can be induced by various agents [e.g., by retinoic acid (RA)] to mature to a nonproliferative end stage. It has also been found that normal mature granulocytes produce a chalone-like "hemoregulatory peptide (HP)" which seems to be involved in the inhibitory proliferation control of myelopoietic cells. In view of the intended use of maturation induction treatment as an alternative to current antileukemic therapy it appeared to be of interest to know if granulocytes, obtained by RA treatment of the promyelocytic leukemia cell line HL-60, would produce normal HP or if their transformed phenotype would cause production of deviant regulatory peptide(s). It was found that conditioned media from RA-treated HL-60 cells inhibited myeloid proliferation but strongly stimulated the growth of erythroid and lymphoid cells. A low molecular weight thiol-containing peptide was isolated which inhibited colony formation by normal granulocyte-macrophage committed stem cells but unlike HP had no effect on (untreated) HL-60 cells themselves. It was also shown that the HL-60 RA peptide is chemically different from HP in terms of molecular size, electrophoretic mobility, composition, and NH2-terminal sequence, which was determined as glutamine-aspartic acid-proline. It is concluded that differentiated HL-60 cells produce hemoregulatory factor(s) with properties different from those of normal HP. The implication of a possible abnormal regulatory behavior of induced leukemic populations is discussed with respect to leukemia therapy by differentiation induction.

Glioma cell interactions with fetal rat brain aggregates in vitro and with brain tissue in vivo.

Two malignant rat neurogenic cell lines have been confronted with reaggregation cultures of fetal rat brain in a stationary culture system. In this organ culture system several morphological aspects of the developing brain are imitated. The malignant cell lines showed invasive and progressive growth into the brain tissue, ending with a completely destroyed aggregate within 10 days of coculture. One cell line (BT5C) showed solid invasion with groups of cells replacing different layers of the brain tissue. A considerable degree of normal cell lysis was seen, both at the edge of invasion and at distant parts of the aggregate. This cell line seemed to activate macrophages in the aggregates. In addition, conditioned medium from these cultures caused degeneration of the brain aggregates. The other cell line (BT4Cn) showed invasion by single cells where the replacement and destruction of the tissue was not accompanied by distant lysis. Both cell lines showed the same pattern of invasion in vitro as was seen in the brains of isogeneic animals in vivo, showing that the individual tumor characteristics were also reflected during invasive growth in organ culture.

Immunocytochemical characterization of extracellular matrix proteins expressed by cultured glioma cells.

The immunolocalization of type I, III and IV collagens and fibronectin in two rat glioma cell lines in vitro (BT4C and BT4Cn) is described. In addition, antibodies against denatured type I and III collagens were used to study breakdown products of native type I and III collagens. For the BT4C cells, the extracellular matrix expression in monolayer cultures and in multicellular tumor spheroids was compared. Type IV collagen was strongly expressed in BT4C tumor spheroids but was negative in the corresponding monolayer cultures. Denatured type I collagen was found both in monolayers and in spheroids of BT4C, suggesting either a rapid turnover (i.e., synthesis and immediate breakdown) of type I collagen or an altered collagen gene transcription. Both cell lines were negative for native type I and III and denatured type III collagen. Fibronectin was strongly expressed in both cell lines. Supporting the immunofluorescence data, the hydroxyproline content in the tumor spheroids was twice the amount found in monolayer cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunoblotting also verified the immunostaining experiments, showing that glioma spheroids and injected tumor cells have the potential for fibronectin and collagen production, given the appropriate growth conditions.

Electrophysiological properties of ethylnitrosourea-induced, neoplastic neurogenic rat cell lines cultured in vitro and in vivo.

A comparative analysis was performed on the electrophysiological properties of 11 neoplastic neurogenic cell culture lines and five other cell lines of different origin (HV1C, rat bile duct carcinoma; BICR/M1RK, rat mammary tumor; HeLa, human cervix carcinoma; 3T3, mouse embryo; REe, rat embryo). Neurogenic lines were derived either from N-ethyl-N-nitrosourea-induced neoplasms of the nervous system or from cultured fetal rat brain cells that had undergone neoplastic transformation in vitro after exposure to N-ethyl-N-nitrosourea in vivo. Electrical membrane excitability was lacking in all neurogenic cells analyzed. Their membrane potential and input resistance values were similar to those of the nonneurogenic lines. Intercellular ionic coupling was consistently observed between cells of a fibroblastoid shape or cells bearing multiple cytoplasmic processes (i.e., all neurogenic lines HV1C, BICR/M1RK, and 3T3). Epithelioid cells (i.e., HeLa, REe, an NV1C subpopulation, and a GV1C1 variant) showed no such intercellular communication. In vivo monolayer cultures on glass coverslips were obtained by a modified i.p. diffusion chamber technique. Under these conditions, the cells (with the exception of a glioma-derived cell line) retained the morphological appearance and electrophysiological properties observed in vitro.

DNA synthesis and ploidy in non-Hodgkin's lymphomas demonstrate intrapatient variation depending on circadian stage of cell sampling.

Significant circadian cell cycle variations with a maximal number of cells in S-phase during the night have been found in a series of 24 patients (18 men and 6 women) with histologically established non-Hodgkin's lymphomas. Pathological lymph nodes of a total of 26 patients were punctured and aspirated by fine needle technique every 4 h during a single 24-h time span. Twenty-four patients (92.3%) had Stage III or IV disease. Twelve patients (46.1%) had low grade, 10 patients (38.5%) had intermediate grade, and 4 patients (15.4%) had high grade lymphomas according to the Working Formulation. The samples were analyzed by flow cytometry, and DNA synthesis (S-phase) and ploidy were determined according to circadian stage. The individual mean 24-h S-phase varied from 2.2 +/- 1.2% (mean +/- SD) to 24.0 +/- 3.3%. Within the group of patients with low grade lymphomas, a wide range in mean S-phase from 2.4 +/- 1.2% to 9.2 +/- 2.8% was observed. The percentage variation within each patient between the lowest and highest S-phase as compared to the lowest value (range of change) during the 24-h time span varied from 21 to 353%, with a mean range of change of 128 +/- 19%. When each individual S-phase series was converted to percent of mean and combined for analysis by one-way analysis of variance to test for time-effect across 2 12-h time spans (8 p.m.-8 a.m. versus 8 a.m.-8 p.m.), S-phase variation according to circadian stage was found to be statistically significant (P < 0.004), with higher values found in the 8 p.m.-8 a.m. time span. By single cosinor analysis, S-phase yielded a near significant P value of 0.069 for the least-squares fit of a 24-h cosine to all data as percent of mean, with the acrophase found to be near midnight (0.05 h). For those patients with low and intermediate grade lymphomas and with mean S-phase values < 10.0%, we found that mean S-phase was higher during winter (5.8 +/- 0.4%) than during spring (3.8 +/- 0.3%) or during fall (3.6 +/- 0.3%) (P < 0.001, analysis of variance). Twenty-one of the 26 patients (80.8%) had an aneuploid, hypodiploid, or near diploid population in one or several of the repeated samples. For the whole series, the DNA indices for the aneuploid populations varied from 1.09 to 1.96, the median DNA index being 1.20.(ABSTRACT TRUNCATED AT 400 WORDS)

Prevention of hematotoxic side effects of cytostatic drugs in mice by a synthetic hemoregulatory peptide.

The application of certain cytostatic drugs causes the recruitment of pluripotent hemopoietic stem cells (CFU-S) into active proliferation. Further application of the drug(s) may then lead to severe and long lasting disturbances of hemopoiesis. We investigated if the hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys (HP5b) could be used to inhibit stem cell recruitment and consequently to protect mice against the toxicity of repeated high doses of 1-beta-D-arabinofuranosylcytosine (ara-C). CFU-S recruitment (induced by injecting a single dose of 900 mg/kg ara-C) was prevented by either treating the bone marrow of these mice in vitro with 1 x 10(-7) M/liter HP5b, or by injecting 0.6 microgram HP5b (10(-9) mol, 30 micrograms/kg) at -2, +2, and +6 h relative to the ara-C injection. Multiple high dose ara-C applications (4 x 900 mg/kg at 0, 7, 24, and 30 h) lead to proliferative activation of CFU-S and resulted in the death of 90% of the mice within 7-9 days. Reconstitution of the hemopoietic system by a bone marrow transplant given after ara-C application decreased the mortality to about 45%, indicating the nonhematological component of ara-C toxicity. A single injection of HP5b (30 micrograms/kg at 26 h, when few CFU-S were found in S phase) decreased the mortality to 59%, not significantly different from the transplanted group. Inactive peptides given instead of HP5b had no protective effect. HP5b did not change the ara-C sensitivity of transformed cell lines (HL-60, Raji, Friend), even not in such cases (myeloid cell lines) where it had a direct inhibitory effect on the cells (e.g., HL-60). These results suggest that HP5b may be used as a myeloprotector in cancer chemotherapy by keeping hemopoietic stem cells out of cycle during the most hazardous treatment phase. Its lack of species specificity, its low toxicity, its high selectivity for hemopoiesis, the small size, as well as the availability through standard synthetic techniques may be of advantage for its clinical use.

Effect of epidermal growth factor on glioma cell growth, migration, and invasion in vitro.

Effects of epidermal growth factor (EGF) and an antibody (Ab-528) reactive against the binding site for EGF on human EGF receptors were studied on multicellular tumor spheroids obtained from three human glioma cell lines with high (D-37 MG), medium (D-247 MG), and low (D-263 MG) levels of EGF receptor expression. The D-247 MG and D-263 MG spheroids grew slowly or not at all in the absence of EGF, while in the presence of EGF they were growth stimulated. Tumor cell migration, as measured by the spread of cells from spheroids on a plastic substratum, was increased by the addition of EGF for all three cell lines. Stimulation of migration could be blocked by a subsequent addition of Ab-528 to the medium at a concentration of 50 micrograms/ml. Invasiveness of glioma cell spheroids into fetal rat brain aggregates was related to EGF receptor expression; the two lines with medium to high receptor expression (D-247 MG and D-37 MG) were invasive, while the line with low EGF receptor expression (D-263 MG) was noninvasive, as assessed by an in vitro coculture assay. In the D-247 MG cell line, morphometry revealed EGF-enhanced invasiveness of the tumor cells. The addition of the Ab-528 to EGF-treated cocultures reduced invasion in both D-247 MG and D-37 MG cell lines. Antibody Ab-528 alone did not affect glioma cell growth or migration but did inhibit invasiveness. The present study suggests that, in brain tumors with an increased number of normal-sized Mr 170,000 EGF receptors, EGF or an EGF-like ligand such as transforming growth factor-alpha may selectively facilitate expansive tumor growth and tumor cell invasion. This effect may in part be blocked or retarded by specific antibodies to the EGF receptor.

Characterization of cyclic adenosine 3':5'-monophosphate-dependent protein kinase isozymes in normal and neoplastic fetal rat brain cells.

Fetal brain cells from rats given a transplacental pulse of N-ethyl-N-nitrosourea progressively acquire malignant characteristics and dedifferentiate when grown in vitro. One aspect of this dedifferentiation is a decreased morphological response to cyclic adenosine 3':5'-monophosphate (cAMP). In the present study, we have characterized and compared the isozymes (I, II) of cAMP-dependent protein kinase in fetal brain cells and in the neoplastically transformed, dedifferentiated BT5C glioma cell line. This is a first approach to find the mechanism behind the subresponsiveness of such cells towards cAMP. It is also part of a broader investigation of the cAMP effector system in cells showing various rates of normal and malignant growth. We found the regulatory and catalytic subunits of cAMP-dependent protein kinase to be expressed to a similar degree in both cell types. Sixty % of the enzyme was located in the 30,000 X g supernatant. The glioma cell line had a significantly higher ratio (1.2) between protein kinase I and II than did the normal fetal cells (0.5). This difference in isozyme distribution was not apparent using conventional methods for enzyme separation and detection, the use of specific antibodies being essential for that purpose. Of the chromatographically separated forms (a, b) of protein kinase II, Form IIa was selectively decreased in the glioma cell line. The alterations of the protein kinases in the glioma cell line described above may be of importance for some of the neoplastic properties of these cells. However, the subdued response of such cells towards cAMP is not explained since the concentrations of cAMP or its analogues required for activation of the kinases were similar for the enzymes from normal and neoplastically transformed cells.

Anti-GM2 monoclonal antibodies induce necrosis in GM2-rich cultures of a human glioma cell line.

The effects of four anti-GM2 monoclonal antibodies (DMAb-1, DMAb-2, DMAb-3, and DMAb-5) were studied on spheroid cultures from a human glioma cell line (D-54 MG) that is known to express high levels of GM2. The spheroids developed central necrosis 48 h after antibody exposures at concentrations greater than 6 micrograms/ml. No necrosis was found with antibodies that had been absorbed with GM2 prior to exposure or with unrelated cytotoxic antibodies. Immunohistochemistry showed that the necrosis started shortly after the antibodies were evenly distributed throughout the spheroids. Light and transmission electron microscopy revealed that a small portion of the cells, mainly in the periphery of the spheroids, was unaffected by antibody exposure. New monolayer cultures established from antibody-treated cells expressed a 50% lower GM2 content as shown by flow cytometry and determination of ganglioside content throughout at least 12 passages. Thus, the GM2-rich D-54 MG cell line has subpopulations of cells with lower GM2 content. Spheroids obtained from this subpopulation developed only minor necrosis after antibody treatment. These results show that GM2 antibodies cause severe necrosis of GM2-containing glioma cells in vitro, but the effect depends on the concentration of antigen, and a threshold number of GM2 molecules is required.

Circadian variations in mouse bone marrow.

Circadian variations in mouse hemopoiesis were investigated in the early spring and summer using spleen colony assay, agar colony technique and differential counting. The multipotent stem cells and committed stem cells as well as the numbers of myelopoietic cells/femur underwent strong circadian variations with similar type of rhythmicity. In both types of stem cells (CFUS, CFUC) the rhythm was correlated to the variations in the numbers of myeloid cells/femur. Highest value (acrophase) was found at 1300 (MET). The amplitudes for CFUS and CFUC were between 50 and 80% of the 24 h mean values (mesor), whereas the variations in the other compartments of the bone marrow were less pronounced. For the CFUC the circadian rhythms were reproducible both at two different times of the year and over 48 h. Changes were found in erythroid cells as well, but the rhythm was not so pronounced.

Inhibitory effects of a synthetic pentapeptide on hemopoietic stem cells in vitro and in vivo.

A synthetic analogue of a pentapeptide associated with mature granulocytes has been investigated for biological effects on stem cell activity in vitro and in vivo. When tested on bone marrow cells from female C3H mice, a short incubation in vitro in doses from 10(-9) to 10(-5) M inhibited myelopoietic colony formation (CFU-C). A maximum of 80% reduction of colony yield was found at 10(-7) M. An oxidized form of the molecule had a stimulatory effect on colony formation, but the inhibitory effect was restored by treatment with a reducing substance (mercaptoethanol). The peptide was nontoxic during continuous exposure of liquid cultures of bone marrow cells for up to 24 h at a dose range of 10(-11) to 10(-4) M. When injected into mice, a dose-dependent inhibitory effect on CFU-C was seen. Maximal effect was obtained by continuous infusion of 1.4 micrograms/h for six days, where only one-fifth of the normal CFU-C number per femur could be retrieved. Prolonged exposure to the same dose level resulted in less reduction. A significant, but less pronounced reduction of spleen colony formation (CFU-S) per femur was seen in the same dose range. Inhibition of both CFU-S and CFU-C was in all cases reversible and mostly accompanied by an overshoot of up to 75% above normal level. In addition, primarily noninhibitory doses led to a secondary increase in the numbers of CFU-S. The total cell number per femur was moderately but significantly reduced, and a prolonged reduction of granulocyte numbers in peripheral blood resulted. No direct toxic effects were seen in vivo on 271 mice given up to 9 mg of the peptide. The results indicate that the peptide may have a regulatory function for stem cell activity in vivo.

Inhibition of phagocytosis by monoclonal antibodies to human myeloid differentiation antigens.

The influence of eight antimyeloid monoclonal antibodies on human leukocyte phagocytosis was investigated using flow cytometry. A granulocyte-specific monoclonal antibody, VIM-D5, inhibited the phagocytosis of both zymosan particles and Staphylococcus aureus in a dose-dependent fashion. In the presence of 5 micrograms/ml, the numbers of phagocyte-associated zymosan particles and bacteria were reduced by about 35% and 40%, respectively. Another monoclonal antibody, VIM-12, reacting with granulocytes, monocytes, and null lymphocytes, inhibited both granulocyte and monocyte phagocytosis of S. aureus. The inhibition was dose dependent, and in the presence of 10 micrograms/ml, the number of phagocyte-associated bacteria was reduced by about 40%. VIM-12 did not influence the phagocytosis of zymosan particles. Both VIM-D5 and VIM-12 inhibited the internalization phase of phagocytosis, whereas the attachment to the phagocyte surface was unaltered. The combined effect of VIM-D5 and VIM-12 was additive, amounting to about 70% reduction of phagocytosis of bacteria. The remaining six antimyeloid antibodies had no effect on leukocyte phagocytosis. The combined use of antimyeloid monoclonal antibodies and flow cytometry appears to be a promising tool for the study of phagocyte functions.