The perturbed cell proliferation kinetics of the EMT6/M/AC tumour in mice treated with anti-mouse lymphocyte serum.

The cell proliferation kinetics of the EMT6/M/AC tumour were determined at two volumes, 6-3 mm3 and 180 mm3, in mice treated with anti-mouse lymphocyte serum, AMLS. Comparison of the growth curve with that obtained in non-AMLS treated animals showed a marked increase in the growth rate at all volumes in the treated group. In contrast, the cell cycle time and the intermitotic phase times were not significantly different in the treated and untreated groups at comparable volumes. The increase in the growth rate in AMLS treated mice was obtained in spite of decreases in both the rate constant for cell production and the growth fraction, and was due to a marked decrease in the rate constant for cell loss.     

THE CELL PROLIFERATION KINETICS OF THE EMT6/M/AC MOUSE TUMOUR AT FOUR VOLUMES DURING UNPERTURBED GROWTH IN VIVO

The cell proliferation kinetics of the EMT6/M/AC mouse tumour were determined at four different volumes between 1–5 mm³ and 175 mm³. The decrease in the growth rate between these volumes was mainly due to a decrease in the rate constant for cell production. A small increase in the rate constant for cell loss occurred, but this was thought to be insignificant. The cell loss factor increased from 40% at 1–5 mm³ to over 70% in the 175 mm³ tumours. An increase in the median cell cycle time, from 14-1 hr to 18-5 hr was also found between these same volumes. Results obtained for the NCTC fibrosarcoma and the R-l rhabdomyosarcoma indicate that there may be a threshold volume in these sarcomas below which little or no cell loss takes place. This was not found in the EMT6/ M/AC tumour.     

The cell proliferation kinetics of the EMT6/M/AC mouse tumour at four volumes during unperturbed growth in vivo.

The cell proliferation kinetics of the EMT6/M/AC mouse tumour were determined at four different volumes between 1-5 mm3 and 175 mm3. The decrease in the growth rate between these volumes are mainly due to a decrease in the rate constant for cell production. A small increase in the rate constant for cell loss occurred, but this was thought to be insignificant. The cell loss factor increased from 40% at 1-5 mm3 to over 70% in the 175 mm3 tumours. An increase in the median cell cycle time, from 14-1 hr to 18-5 hr was also found between these same volumes. Results obtained for the NCTC fibrosarcoma and the R-1 rhabdo-myosarcoms indicate that there may be a threshold volume in these sarcomas below which little or no cell loss takes place. This was not found in the EMT6/M/AC tumour.     

Changes in the extents of viable and necrotic tissue, interstitial fluid pressure, and proliferation kinetics in clone A human colon tumour xenografts as a function of tumour size

Abstract. Total, viable and necrotic tumour tissue, tumour cell yields, and colony forming efficiencies were measured in clone A human colon tumour xenografts as neoplasms grew from about 100 mm³ to about 6000 mm³. The volumes of the total, viable and necrotic compartments were fit using the Verhulst equation to obtain estimates of growth rates and maximal sizes of the various compartments (carrying capacities). Additionally, at four discrete tumour volumes (250, 850, 2500 and 5500 mm³), hypoxic percentages, proportions of parenchymal tumour and host cells, interstitial fluid pressures, and proliferation kinetics including measurements of apoptosis were determined. There were interesting relationships between the shapes of the curves for total, viable and necrotic tissue to some of the other endpoints measured. Specifically, the volumetric growth curves for the total and viable tumour tissue compartments were identical to a volume of approximately 1000 mm³, but diverged at larger sizes, with the viable cell compartment exhibiting a smaller carrying capacity. The shape of the growth curve for the necrotic compartment exactly mimicked that for the total volume compartment, but was delayed in time by about 21 days. Similarity in shape to that of the overall tumour volume/necrotic volume curves was also seen for the curve for the increase in interstitial fluid pressure, and for the increase in the size of the host cell compartment. In contrast, the growth of the hypoxic compartment and of the parenchymal tumour cell compartment were similar in shape to that of the viable compartment. These data indicate that these compartments are functionally linked. Marked changes in cell kinetic parameters occurred as tumour size increased from 250–5500 mm³. The labelling index and growth fractions decreased from 0.256–0.125, and 0.77–0.40 respectively, and the cell loss factor increased from 0.52–0.74. The volumetric and potential doubling times increased from 4.3–17.6 and 2.1–4.6 days respectively. The cell kinetic changes could not be clearly related to the changes in shape of either the overall tumour volume or the viable tumour volume.     

Quantitative studies of superior cervical sympathetic ganglia in a variety of primates including man. II. Neuronal packing density

The volumes of a sample of primate superior cervical sympathetic ganglia were measured and related to body weight and to the number of ganglionic neurons. Estimates of volumes of the ganglia varied between 1.956 mm³ in squirrel monkey and 173.530 mm³ in a human specimen. Average cell densities for the ganglia ranged from 4,455 cells/mm³ in a human ganglion to 32,528 cells/mm³ in a squirrel monkey ganglion. Mean cell territories varied from 0.0000307 mm³ in a squirrel monkey ganglion to 0.0002245 mm³ in a human ganglion. Analysis of the data reveals striking trends of correlation between body size, volume of ganglia, and average cell territories. Since similar correlations have been described for other types of neuronal cell aggregates, it is suggested that for any given nucleus, ganglion or cortical area, the neuronal packing density varies as a function of body size.     

Deficiency of vasodilator-stimulated phosphoprotein (VASP) increases blood-brain-barrier damage and edema formation after ischemic stroke in mice

Background

Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification.

Methodology/Principal Findings

Focal cerebral ischemia was induced in Vasp^−/− mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evan''s Blue tracer was applied to visualize the extent of blood-brain-barrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p<0.05) and edema volumes (1.7 mm³±0.5 mm³ versus 0.8 mm³±0.4 mm³; p<0.0001) were significantly enhanced in Vasp^−/− mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm³±17.3 mm³ versus 39.3 mm³±10.7 mm³, respectively; p<0.01) and a non significant trend (p>0.05) towards worse neurological outcomes.

Conclusion

Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage.     

Human amniotic membrane derived-mesenchymal stem cells induce C6 glioma apoptosis in vivo through the Bcl-2/caspase pathways

High-grade gliomas are difficult to treat. We examined the therapeutic effect of intratumoral administration of human amniotic membrane derived-mesenchymal stem cells (hAMCs) on the growth of gliomas. Tumor volume of the control group was 1632 ± 316 mm³ on day 30, and the group treated with a single intratumoral dose of hAMCs had a tumor volume of 1128 ± 269 mm³ (P < 0.05). Thus, administration of hAMCs significantly reduced tumor size. In rat glioma tissues treated with single and multiple dosages of hAMCs, there was a reduction in tumor volume of approximately 30.9 and 49.5%, respectively. We further evaluated the glioma tissue using Western blotting analysis and observed that the expression of Bax, caspase-8 and caspase-3 were greatly increased and the expression of Bcl-2 was greatly decreased in tumors treated with hAMCs. Sections of nude mice treated with hAMCs clearly showed the presence of an increase in apoptotic cells. The data collected herein confirms for the first time that hAMCs can inhibit C6 glioma growth and induce apoptosis of C6 gliomas in vivo. This demonstrates that hAMCs are a potential new therapeutic agent for the treatment of gliomas.     

CYTOKINETICS OF TUMOUR AND ENDOTHELIAL CELLS AND VASCULARIZATION OF LUNG METASTASES IN C3H/HE MICE

A model of lung metastases was developed using intravenous injection of tumour cell aggregates of spontaneous C3H/He mammary tumours in syngeneic mice. the growth rate of lung tumours decreased with increasing tumour volume, with mean host survival of 46 days. the cytokinetics of individual tumours ranging between 0.004 and 4.2 mm³ in volume were studied. the labelling index (LI) ranged between 12 and 17%, the DNA synthesis time (T_s) being 9–10 hr. the growth fraction (GF) ranged between 26 and 38%. the cell cycle time (T_c) was found to be 18–19 hr. the LI and the GF decreased with increasing tumour volume doubling time (T_d). No correlation was found between the tumour volume and T_c. the LI of endothelial cells within these tumours, ranging between 0.004 and 4.2 mm³ in volume was 14–15% and endothelial cell proliferation was not affected by tumour growth. Vascular parameters were also determined for these tumours as a function of tumour volume. Vascular volume increased with increasing tumour size while the percentage of capillary vessels decreased. the cellular volume to capillary volume ratio increased with increasing tumour volume. Necrosis was observed in 0.27 mm³ tumours and increased with increasing tumour size. The results from these studies suggested that the age-dependent decrease in proliferative activity of tumour cells growing in the lung is related to change in effective vascularity.     

Tumor-derived Fas ligand induces toxicity in lymphoid organs and plays an important role in successful chemotherapy

 Recent studies have suggested that Fas ligand (FasL⁺) tumor cells can induce apoptosis in Fas⁺ T cells. However, the effect of growth of FasL⁺ tumors in vivo, on lymphoid tissues of the host is not clear and therefore was the subject of this investigation. Injection of FasL⁺ LSA tumor caused a significant decrease in cellularity of the thymus and spleen, resulting from marked apoptosis, in syngeneic C57BL/6+/+ (wild-type) but not C57BL/6-lpr/lpr (Fas-deficient) mice. The tumor-induced toxicity resulted from tumor-derived rather than host-derived FasL, inasmuch as LSA tumor growth in C57BL/6-gld/gld (FasL-defective) mice, induced marked apoptosis and toxicity in the thymus and spleen. The LSA tumor growth induced a significant decrease in the percentage of CD4⁺CD8⁺ T cells in the thymus of C57BL/6+/+ mice and an increase in the percentage of CD4⁺, CD8⁺ and CD4⁻CD8⁻ T cells. Of the four subpopulations tested, the CD4⁺CD8⁺ T cells showed maximum apoptosis. The LSA (FasL⁺) but not P815(FasL⁻) tumor cell lysates and culture supernatants induced marked apoptosis in Fas⁺ thymocytes, when tested both in vitro and in vivo. The LSA-tumor-induced apoptosis in vitro was inhibited by antibodies against FasL or by caspase and other inhibitors of apoptosis. Chemotherapy of LSA-tumor-bearing C57BL/6+/+ mice at advanced stages of tumor growth failed to cure the mice, whereas, more than 80% of LSA-tumor-bearing C57BL/6-lpr/lpr mice, similarly treated, survived. Together, the current study demonstrates that FasL produced by LSA tumor cells is functional in vivo and can cause severe toxicity in lymphoid organs of the host. Also, Fas/FasL interactions may play an important role in the successful chemotherapy of FasL-bearing tumor. Received: 31 August 1999 / Accepted: 12 November 1999     

Effects of Toxoplasma gondii and Toxocara canis Antigens on WEHI-164 Fibrosarcoma Growth in a Mouse Model

Cancer is the main cause of death in developed countries. However, in underdeveloped countries infections and parasitic diseases are the main causes of death. There are raising scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of Toxoplasma gondii and Toxocara canis egg antigens in comparison with Bacillus Calmette Guerin (BCG) (known to have anticancer distinctive) on WEHI-164 fibosarcoma transplanted to BALB/c mice was investigated. Groups of 6 male BALB/c mice injected with T. gondii antigen, BCG, or T. canis egg antigen as case groups and alum alone as control groups. All mice were then challenged with WEHI-164 fibrosarcoma cells. The mice were examined for growth of the solid tumor and the tumor sizes were measured every other day up to 4 wk. The mean tumor area in T. gondii, BCG, or alum alone injected mice in 4 different days of measurements was 25 mm², 23 mm², and 186 mm² respectively. Also the mean tumor area in T. canis injected mice in 4 different days was 25.5 mm² compared to the control group (alum treated) which was 155 mm². T. gondii parasites and T. canis egg antigens induced inhibition of the tumor growth in the fibrosarcoma mouse model. We need further study to clarify the mechanisms of anti-cancer effects.     

Ultraviolet-induced mutations to asparagine independence in Jensen sarcoma cells in vitro

UV-irradiation induces an exponential increase in the frequency of mutation from asparagine requirement to asparagine non-requirement in Jensen sarcoma cells grown in vitro. The corrected mutation frequency increases from the spontaneous rate of 5.1·10^?6 per cell to 1248·10^?6 per cell with a dose of 180 erg/mm² of 254 nm UV A substantial increase was oberved even without correction for survivors, and no significant difference was observed in the UV sensitivity of asparagine-requiring and non-requiring Jensen clones. When Jensen cells were plated at low densities in a feeder layer of LMTK-cells inactivated by HAT medium, an increase in the cloning ability of the former was observed as compared to appropriate controls without the feeder layer, but the increase was constant over all doses of UV tested. Revertants are stable and possess measurable asparagine synthetase.It is concluded that UV is an extremely effective mutagen in this system.     

Multi-Modality Therapeutics with Potent Anti-Tumor Effects: Photochemical Internalization Enhances Delivery of the Fusion Toxin scFvMEL/rGel

Background

There is a need for drug delivery systems (DDS) that can enhance cytosolic delivery of anti-cancer drugs trapped in the endo-lysosomal compartments. Exposure of cells to specific photosensitizers followed by light exposure (photochemical internalization, PCI) results in transfer of agents from the endocytic compartment into the cytosol.

Methodology and Principal Findings

The recombinant single-chain fusion construct scFvMEL/rGel is composed of an antibody targeting the progenitor marker HMW-MAA/NG2/MGP/gp240 and the highly effective toxin gelonin (rGel). Here we demonstrate enhanced tumor cell selectivity, cytosolic delivery and anti-tumor activity by applying PCI of scFvMEL/rGel. PCI performed by light activation of cells co-incubated with scFvMEL/rGel and the endo-lysosomal targeting photosensitizers AlPcS_2a or TPPS_2a resulted in enhanced cytotoxic effects against antigen-positive cell lines, while no differences in cytotoxicity between the scFvMEL/rGel and rGel were observed in antigen-negative cells. Mice bearing well-developed melanoma (A-375) xenografts (50–100 mm³) were treated with PCI of scFvMEL/rGel. By 30 days after injection, ∼100% of mice in the control groups had tumors>800 mm³. In contrast, by day 40, 50% of mice in the PCI of scFvMEL/rGel combination group had tumors<800 mm³ with no increase in tumor size up to 110 days. PCI of scFvMEL/rGel resulted in a synergistic effect (p<0.05) and complete regression (CR) in 33% of tumor-bearing mice (n = 12).

Conclusions/Significance

This is a unique demonstration that a non-invasive multi-modality approach combining a recombinant, targeted therapeutic such as scFvMEL/rGel and PCI act in concert to provide potent in vivo efficacy without sacrificing selectivity or enhancing toxicity. The present DDS warrants further evaluation of its clinical potential.     

Repression of folate synthesis in the logarithmic phase of Euglena gracilis growth

Microbiological assay showed that in Euglena gracilis cultures the amount of cell folates reaches its maximum at the beginning of the culture cycle and rapidly and markedly decreases long before the cells reduce their duplication rate. [³H]Folic acid was a suitable precursor of Euglena folates (a full recovery of growth in sulfanilamide inhibited cultures was obtained by addition of folic acid), and a complete radiochromatographic profile of cell folates was obtained by separation on G-25 columns. This allowed the measurement of the rate of folate degradation, obtained from the rate of radioactivity disappearance in chromatographic patterns of extracts corresponding to increasing times of culture cycles. With the exception of the stationary phase, the process of folate degradation showed a first order kinetics with a rate constant of 5.4 + 10⁻⁴ min⁻¹ and a half-life of 21 h and 12 min. The rate of folate biosynthesis was calculated by adding the amount of degradation to the measured increase (or decrease) in cell folates. The specific rate reached its maximum (8.6 ng of folinic acid equivalent h⁻¹ for 10⁶ cells) as the culture entered the logarithmic phase of growth and rapidly decreased to about 1/20 of this value before leaving it. This indicates that the logarithmic phase of growth corresponds to a phase in which folate biosynthesis is strongly repressed.     

Light/Dark Cycle and Temperature-Their Impact on Phosphate-Limited Growth of Oscillatoria redekei VAN GOOR in Semicontinuous Culture

Phosphate-limited growth of Oscillatoria redekei in semicontinuous culture has been studied under conditions of continuous illumination at 20 °C as well as in a 12/12 hours light-dark cycle at temperatures between 5 °C and 20 °C. The subsistence quota (q₀) amounted to 0.052 μmol mm⁻³ under all conditions, when the phosphate quota was expressed on the basis of cell volume. The interaction between temperature and phosphate quota and its impact on growth rate are described by the following model: Parameter values are t_opt=24.5 °C, t_min=0.95 °C, μ_max =0.873 d⁻¹. The maximum phosphate quota was found to depend on temperature and to increase along with declining temperature.     

CELL POPULATION KINETIC PROFILE OF THE MOUSE THYMUS, AND THE CHANGES INDUCED BY PREDNISOLONE

The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [³H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G₁. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [³H]TdR techniques.     

The influence of CO2 enrichment, phosphorus deficiency and water stress on the growth, conductance and water use of Pinus radiata D. Don

Abstract. Seedlings of Pinus radiata D. Don were grown in growth chambers for 22 weeks with two levels of phosphorus, under either well-watered or water-stressed conditions at CO₂ concentrations of either 330 or 660mm³ dm^?3. Plant growth, water use efficiency and conductance were measured and the relationship between these and needle photosynthetic capacity, water use efficiency and conductance was determined by gas exchange at week 22. Phosphorus deficiency decreased growth and foliar surface area at both CO₂concentrations; however, it only reduced the maximum photosynthetic rates of the needles at 660 mm³ CO₂ dm^?3 (plants grown and measured at the same CO₂ concentration). Water stress reduced growth and foliar surface area at both CO₂ concentrations. Increases in needle photosynthetic rates appeared to be partly responsible for the increased growth at high CO₂ where phosphorus was adequate. This effect was amplified by accompanying increases in needle production. Phosphorus deficiency inhibited these responses because it severely impaired needle photosynthetic function. The relative increase in growth in response to high CO₂ was higher in the periodically water-stressed plants. This was not due to the maintenance of cell volume during drought. Plant water use efficiency was increased by CO₂ enrichment due to an increase in dry weight rather than a decrease in shoot conductance and, therefore, transpirational water loss. Changes in needle conductance and water use efficiency in response to high CO₂ were generally in the same direction as those at the whole plant level. If the atmospheric CO₂ level reaches the predicted concentration of 660 mm³ dm^?3 by the end of next Century, then the growth of P. radiata will only be increased in areas where phosphorus nutrition is adequate. Growth will be increased in drought-affected regions but total water use is unlikely to be reduced.     

Secretion rates and levels of vascular endothelial growth factor in clone A or HCT-8 human colon tumour cells as a function of oxygen concentration

Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growth factor (VEGF) mRNA expression is up-regulated and VEGF protein is concomitantly increased. To establish a quantitative relationship between VEGF protein levels and oxygenation, we exposed exponentially growing clone A or HCT-8 human colon tumour cells in vitro (22 h at 37°C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme-linked immunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT-8 cells exposed to either air (21% O₂) or the 0.01% O₂ mixture respectively increased from about 73 to 1270, and 1.5 to 1180 pg/10⁶ cells (about 17- and 80-fold increases). The shapes of the response curves (log of the intracellular VEGF concentrations v. log oxygen concentration) for both cell types were sigmoidal. However, intracellular VEGF levels in HCT-8 cells were always less than that of clone A cells until levels of about 0.3 to 0.1% O₂ were reached. Levels of VEGF in the supernatant were also increased after the 22 h hypoxic exposures. Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lines as a function of oxygen percentage. For clone A cells, the secretion rate (pg/10⁶ cells/h) in 21% O₂ was 62.5. This rate increased to 428.8 pg/10⁶ cells/h at 0.01% O₂, a 7-fold increase. For HCT-8 cells, levels in the medium at 21% O₂ were too low to be measured by ELISA. However, between 10% and 0.01% O₂, secretion rates increased from 5.0 to 376.0 pg/10⁶ cells/h, a 75-fold increase. Therefore, at very low O₂ levels, VEGF secretion rates were similar in the two cell lines. We propose that the different VEGF responses of clone A and HCT-8 colon tumour cells to hypoxic stress in vitro are related to the in vivo observation that the respective hypoxic percentages of solid neoplasms originating from these cell lines are markedly different (i.e. about 3 versus 80%) at equivalent volumes of 750 mm³.     

Cell arrangement,mass and dimension ofStreptococcus faecalis during growth

During exponential growth ofStreptococcus faecalis, the distribution of cell arrangements remains constant, but depends on the growth rate. The predominant cell arrangements are diplococci (about 60–80% of total cells) the amount of which varies only little with the growth rate. A clear correlation exists for cells growing as chains; the amount decreases from about 20% at μ=2.0 to about 6% at μ=0.45. After cessation of growth in the stationary phase, the number of diplococci and chains decreases and the number of monococci increases; after 10 h in the stationary phase, more than 50% of the cells have become monococci. The dry weight of 2.5×10⁻¹⁰ mg/cell remains constant at different growth rates, while cell size shows small differences on different growth media. Treatment of exponentially growing cultures with crystal violet or nitrofurantoin results in faster sedimentation on sucrose gradients of treated cultures compared to untreated cultures. While crystal violet effects an increased chain formation, treatment with nitrofurantoin results in an increase of the size of the individual cell.     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.     

Ki-67 (MIB 5) immunostaining of mouse lung tumors induced by 4-nitroquinoline 1-oxide

 We immunostained mouse lung tumors using a mouse monoclonal antibody against recombinant Ki-67 antigen (clone; MIB 5) to establish an MIB 5 immunostaining method and to determine the extent of MIB 5 labeling to monitor cell proliferation activity in mouse lung tumors. A/J mice, treated with 4-nitroquinoline 1-oxide, were killed after 18 months. One hour before killing, bromodeoxyuridine (BrdU) was injected intraperitoneally. Lung tissues including tumors were fixed with phosphate-buffered 4% paraformaldehyde and embedded in paraffin. For MIB 5 immunostaining, two antigen-retrieval buffers, citrate buffer pH 6 and TRIS-HCl buffer pH 9.5 containing 5% urea, were tested, and constant and reproducible staining was obtained only with the TRIS-HCl buffer. The mean values of the MIB 5-positive cell index (PCI), the BrdU labeling index (LI), and the mitotic cell count for adenocarcinomas were 4.6%, 2.3%, and 7/mm², and those for adenomas were 1.2%, 0.7%, and 1.3/mm², respectively. Each of these values was significantly higher for adenocarcinomas than for adenomas. A close correlation was seen between the MIB 5 PCI and the BrdU LI for adenocarcinomas and adenomas and between the MIB 5 PCI and the mitotic cell count in adenocarcinomas. Thus, MIB 5 immunostaining is a useful method for assessing the proliferative activity of mouse tumor tissues. Accepted: 23 June 1998