Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line

The effect of irradiation depends on the oxygenation status of the tissue, while irradiation itself also changes the oxygenation and perfusion status of tissues. A better understanding of the changes in tumor oxygenation and perfusion over time after irradiation will allow a better planning of fractionated radiotherapy in combination with modifiers of blood flow and oxygenation. Vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (pimonidazole) were studied in a human laryngeal squamous cell carcinoma tumor line grown as xenografts in nude mice. The effect of a single dose of 10 Gy X rays on these parameters was evaluated from 2 h to 11 days after irradiation. Shortly after irradiation, there was an 8% increase in perfused blood vessels (from 57% to 65%) followed by a significant decrease, with a minimum value of 42% at 26 h after irradiation, and a subsequent increase to control levels at 7 to 11 days after irradiation. The hypoxic fraction showed a decrease at 7 h after treatment from 13% to 5% with an increase to 19% at 11 days after irradiation. These experiments show that irradiation causes rapid changes in oxygenation and perfusion which may have consequences for the optimal timing of radiotherapy schedules employing multiple fractions per day and the introduction of oxygenation- and perfusion-modifying drugs.     

Isolation and characterization of 5'-nucleotidase of a human pancreatic tumor cell line

5'-Nucleotidase of a human pancreatic tumor cell line (PaTu II) has been purified to homogeneity after extraction with detergent followed by two affinity chromatographic steps. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified 5'-nucleotidase revealed a single polypeptide band of 67 kDa. The Western blotted enzyme can be overlaid with concanavalin A proving its glycoprotein nature. After treatment with endoglycosidase F the deglycosylated 5'-nucleotidase exhibits an apparent molecular mass of 58 kDa. The kinetic properties of the solubilized enzyme have been determined (Km (AMP) of 4.0 microM; Vmax (AMP) = 8.6 muMOL/min.mg). Adenosine 5'-[alpha,beta-methylene]diphosphate is a competitive inhibitor of 5'-nucleotidase, whereas concanavalin A inhibits the enzymatic activity in a non-competitive manner. Polyclonal antibodies against purified 5'-nucleotidase of PaTu II have been produced which inhibit its enzymatic activity. Polyclonal antibodies raised against the enzyme purified from rat liver or bull seminal plasma also recognize 5'-nucleotidase of PaTu II cells, whereas polyclonal and monoclonal antibodies against the enzyme derived from chicken gizzard show no cross-reactivity. 5'-Nucleotidase appears to be concentrated in the plasma membrane of PaTu II cells as judged by cell fractionation and indirect immunofluorescence studies.     

Radiobiological effects of total body irradiation on the spinal cord

Total body Irradiation (TBI) is often used for conditioning, prior to bone marrow transplantation. Doses of 8–14 Gy in 1–8 fractions over 1–4 days are administered using low dose rate external beam radiotherapy (EBRT). When necessary, consolidation EBRT using conventional doses, fractionation and dose rate is given. The irradiated volume usually contains critical organs such as spinal cord. The purpose of this study was to assess the biologic effect of TBI on the spinal cord in terms of EQD₂ (equivalent dose given in fractions of 2 Gy). EQD₂ values were calculated using the linear-quadratic generalized incomplete repair (IR) model that incorporates IR between fractions and low dose rate irradiation corrections and accounts for mono and bi-exponential repair. Three fractionation schemes were studied as function of dose rate: 8 Gy in 1 and 2 fractions and 12 Gy in 8 fractions. For the 12 Gy in 8 fractions scheme, the influence of dose rate on EQD₂ was limited because the effect of IR between fractions dominates. For the 8 Gy in 1 fraction scheme, significant sparing of the spinal cord may be achieved for low dose rate (5–20 cGy/min). The extent of effects depends on the parameters used. The IR model provides a useful mathematical framework for examination of the effects of fractionated treatments of varying dose rate. Reliable experimental data are needed for accurate assessment of radiation damage to the spinal cord following fractionated low dose rate TBI.     

Incorporation of acylated antibody into planar lipid multilayers: characterization and cell binding

Multiple (up to 14) layers of lipid were deposited onto an alkylated glass surface by dialysis of egg phosphatidylcholine (PC) and deoxycholate mixed micelles in the presence of alkylated glass coverslips. The amount of lipid associated with the coverslips was measured by using radioactive PC. It was found that the number of PC molecules in the multilayer increased with increasing initial lipid concentration in the dialysis mixture. Inclusion of cholesterol resulted in a significant increase in the amount of total lipid deposited in the multilayer. However, the PC/cholesterol ratio was up to 2-fold higher in the multilayers than in the liposomes present in the same dialysis bag. In addition, mouse monoclonal anti-H2Kk antibody which had previously been derivatized with palmitic acid could be readily incorporated into the lipid multilayer during dialysis. Measurements of lateral mobility with the fluorescence recovery after photobleaching technique on fluorescently labeled lipid or antibody in the multilayer showed that the lipid molecules diffused rapidly while the antibodies were essentially immobile. Lymphoma cells such as RDM4 cells expressing surface H2Kk glycoproteins could rapidly bind to the antibody-containing multilayers. The binding was blocked by free antibody or by goat anti-mouse immunoglobulin G, indicating the immunospecificity of the binding. Cell binding to the multilayer also exhibited a threshold dependence on the antibody density of the multilayer. A lower threshold was found for cells expressing a higher surface density of H2Kk. This system may be useful for model studies of cellular recognition.     

Yeast mitochondrial DNA characterization after ultraviolet irradiation

Yeast mitochondrial (mtDNA) ³H-labelled was isolated from exponential phase cells after ultraviolet light irradiation. Both the size and amount of mtDNA were found to be reduced during a 40-h liquid-holding (LH) period in non-growth medium following irradiation as compared to the mtDNA recovered from nonirradiated cells under similar conditions. After the LH period, previously irradiated cells were suspended in growth medium containing [¹⁴C]adenine. Double labelled mtDNA (³H and ¹⁴C) was isolated from cells samples removed during new growth. A recovery in the amount and size of mtDNA was observed in irradiated cells during new growth. These biochemical studies agree with the observed loss and recovery of mtDNA genetic markers in UV-irradiated exponential phase yeast after a period of LH and new growth resp.     

Establishment and characterization of a human ovarian granulosa tumor cell line (HSOGT)

We successfully established a novel ovarian granulosa tumor cell line (HSOGT). The tumor tissue of the ovary was derived from a 25 year-old Japanese woman under her consent. The cell line was maintained for over 14 months, subcultured more than 73 times, and had a population doubling time of 18.9 hours. Phase contrast microscopy displayed a pavement-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy and the mode was 83; many marker chromosomes were observed. The HSOGT was also successfully xenotransplanted into nude mice. The cell line produced estradiol and has preserved some characters of granulosa cells with stable growth in vitro. We firmly believe that this cell line will be a most useful tool for endocrinological investigation of human granulosa cells.     

Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids

Reports of spontaneous regressions of metastases and the demonstration of tumor-reactive cytotoxic T lymphocytes indicate the importance of the host's immune system in controlling the devastating course of metastatic renal cell carcinoma. Recent research indicates that immunization with hybrids of tumor and antigen presenting cells results in protective immunity and rejection of established tumors in various rodent models. Here, we present a hybrid cell vaccination study of 17 patients. Using electrofusion techniques, we generated hybrids of autologous tumor and allogeneic dendritic cells that presented antigens expressed by the tumor in concert with the co-stimulating capabilities of dendritic cells. After vaccination, and with a mean follow-up time of 13 months, four patients completely rejected all metastatic tumor lesions, one presented a 'mixed response', and two had a tumor mass reduction of greater 50%. We also demonstrate induction of HLA-A2-restricted cytotoxic T cells reactive with the Muc1 tumor-associated antigen and recruitment of CD8+ lymphocytes into tumor challenge sites. Our data indicate that hybrid cell vaccination is a safe and effective therapy for renal cell carcinoma and may provide a broadly applicable strategy for other malignancies with unknown antigens.     

Analysis of cell survival after multiple fractions per day and low-dose-rate irradiation of two in vitro cultured rat tumor cell lines

Two rat tumor cell lines which differ significantly in radiosensitivity, a rhabdomyosarcoma (R-1) and a ureter carcinoma (RUC-2), were treated with multiple fractions per day and low-dose-rate gamma radiation. The purpose of these experiments was to investigate (i) the influence of fraction size and interfraction interval on repair of sublethal damage (SD) and (ii) whether low-dose-rate irradiation can be simulated by giving multiple fractions per day which might be applied in clinical treatments. In both cell lines, multiple doses were given at 1- to 4-hr intervals. SD repair was at a maximum in 2 hr but did not reach the theoretically expected level. For both cell lines, survival at higher total doses was different from that theoretically expected if repair of SD was assumed to be completed and at the maximum level. To account for the observation that less than complete repair of SD occurred, theoretical survival curves were calculated with the assumption of a constant but less than 100% level of SD repair. Experimental data correlated well with these calculated curves. There were only very small differences in survival after the different multiple fractions per day regimens. Survival after irradiation at a dose rate of 1.00 Gy/hr was found to be similar to that after multiple fractions per day.     

Radiobiological properties of human hematopoietic and stromal marrow cells

Effective hematopoiesis requires the presence of normal hematopoietic progenitors and a supporting microenvironment. Impairment of one of these marrow compartments will result in marrow failure. Total body irradiation (TBI) followed by bone marrow transplantation (BMT) is becoming an established modality in the treatment of malignant hematopoietic disorders. The objectives of irradiation are to ablate host marrow and immunocompetent cells as well as to eradicate neoplastic cells. Although leukemic cells are thought to have the same radiobiological characteristics as their normal counterparts, it has been proposed recently that some leukemic cells may possess a substantial capacity to repair sublethal radiation damage. Thus, radiation administered at different dose rates or fractions might differ in its ability to ablate malignant cells and consequently affect the relapse rate in the post-transplant period. Different modes of irradiation can also affect the proliferative capacity and the hematopoietic supportive function of the marrow microenvironment. Bone marrow ablation must be accomplished with the least possible damage to other tissues. Impairment of the proliferative capacity of the marrow microenvironment or its hematopoietic supportive function can result in graft failure in the post-transplant period. In this review, we discuss the radiobiological characteristics of normal hematopoietic, leukemic and stromal cells and their relevance to bone marrow transplantation.     

Fractionated irradiation can induce functionally relevant multidrug resistance gene and protein expression in human tumor cell lines

The molecular basis of radiotherapy-related multidrug resistance (MDR) is still unclear. Here we report on a study investigating the effect of fractionated irradiation on expression of the MDR-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP), the respective mRNAs, and the functional consequences. Cells of six colon and five breast cancer cell lines were irradiated with a total dose of 27 Gy, five fractions of 1.8 Gy per week. The mRNA expression was measured by quantitative RT-PCR, protein levels and drug sensitivity to cisplatin, doxorubicin and bendamustine were assessed by flow cytometry. Breast cancer cell lines showed enhancement of the mRNAs encoding for P-gp, MRP1 and LRP in comparison to nonirradiated cells. No up-regulation of the three mRNA species was observed in the colon cancer cell lines. After irradiation, three breast cancer cell lines showed an up-regulation of LRP, one line an up-regulation of MRP1, and four lines a small up-regulation of P-gp. In the colon cancer cell lines, radiation induced significant enhancement of all three proteins. In comparison to controls, the irradiated cells lines showed a significant resistance to cisplatin, doxorubicin and bendamustine. This study confirms the prior reports of enhancement of P-gp and MRP1 after irradiation, which is accompanied by a multidrug resistance phenomenon, but in addition proposes a novel mechanism in the appearance of MDR after radiation-induced enhancement of LRP.     

Noise amplification in human tumor suppression following gamma irradiation

The influence of noise on oscillatory motion is a subject of permanent interest, both for fundamental and practical reasons. Cells respond properly to external stimuli by using noisy systems. We have clarified the effect of intrinsic noise on the dynamics in the human cancer cells following gamma irradiation. It is shown that the large amplification and increasing mutual information with delay are due to coherence resonance. Furthermore, frequency domain analysis is used to study the mechanisms.     

Establishment and characterization of a new human germ cell tumor strain (NOC-Ts) in culture

We have derived and characterized a new cell line from a teratoma with a embryonal carcinoma and seminoma. The medium used for the cell culture was Eagle's MEM synthetic culture medium (Gibco Inc.) supplemented with 10% new calf serum (Tissue plus, Mitsubishi Kasei Co.). Subcultures were performed on 3 to 4 days basis at 1:2 split by the use of 0.25% trypsin solution. The morphology of obtained cells was a epithelial-like shape and the cell grew in a monolayered sheet with about 30-32 hrs of population doubling time. The model chromosome number of this cell line was 73 including one large submetacentric marker chromosome. The temperature sensitivity and the tumorigenicity of this cell were tested in this experiment.     

Purification and characterization of a unique human interleukin 1 from the tumor cell line U937

Interleukin 1 (IL 1) produced by a human tumor cell line was purified to homogeneity by a three-step chromatographic method and was tested in various assays for multiple biologic properties. The purified IL 1 stimulated the proliferative response of the D10.G4.1 cell line, a mouse IL 1 indicator T cell; caused the release of prostaglandin E2 and prostacyclin from cultured human foreskin fibroblasts and from primary human umbilical vein endothelial cells; and elicited characteristic endogenous pyrogen fever in rabbits. To stimulate IL 1 production, the histiocytic lymphoma cell line U937 was incubated with the exotoxin from toxic shock strains of Staphylococcus aureus. Supernatants from stimulated U937 cells were concentrated, and were applied to a reverse-phase HPLC column. IL 1 activity was eluted from the column at high acetonitrile concentration. Subsequent chromatography over hydroxyapatite yielded a single IL 1 species with a pI of 5.5. IL 1 was then purified to homogeneity by gel exclusion HPLC migrating as a 14 kDa species. The molecular size was confirmed by SDS-PAGE and was visualized as a single molecule by silver staining; biologic activity was recovered from the same region of the gel. Limited N-terminal sequence analysis suggested some homology to the pI 7 form of the human blood monocyte IL 1. The pI 5.5 IL 1 produced by U937 cells was only partially neutralized with anti-human monocyte IL 1 antibody, suggesting that U937-derived IL 1 is structurally related to one of the molecularly cloned IL 1 species. IL 1 from stimulated U937 cells possesses the functional characteristics of monocyte IL 1 but may represent a structurally unique IL 1 species, as determined by sequence analysis, size, and antibody reactivity.     

Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.     

Analysis of cell kinetics using BrdU monoclonal antibody on cultured tumor cells after irradiation and treatment with anti-cancer drugs

Flow cytometry (FCM) permits instantaneous determination of the percentages of cells in various phases of cell cycle using BrdU-PI double staining method, and allowing rapid evaluation of the effects of irradiation and anti-cancer drugs (ACNU, ADR, BLM) on the cell kinetics. In this study, the growth inhibition and changes in the cell kinetics after irradiation and chemotherapy were examined according to the growth curve analysis and BrdU-PI method to evaluate the usefulness of BrdU-PI method for assessment of the effect of the treatments. By the conventional method based on the DNA histogram, accurate determination of S cell fraction was difficult due to overlapping of the DNA contents of G1 cells and early S cells and those of late S cells and G2 cells. BrdU-PI double staining allowed direct differentiation of G1, S, and G2 + M cells, especially between G1-S and S-G2 + M cells. The analysis of cell kinetics using BrdU is advantageous in comparison to the conventional autoradiographic methods because it allows more rapid assay with very high sensitivity. By the present BrdU method, rapid transition to the G1-S phase was observed within 4 hours after exposure to radiation and anti-cancer drugs. This initial G1 arrest induced by irradiation was confirmed for the first time by the present BrdU-PI double staining. The present method is considered to be indispensable for evaluation of the percentage of S cells in the tumor tissue and analysis of cell kinetics after irradiation and chemotherapy against cancer.     

Structural characterization of a rat acinar cell tumor

A transplantable acinar cell tumor of the rat pancreas has been examined by light and electron microscopy. The tumor cells, though highly cytodifferentiated and characterized by the presence of abundant rough-surfaced endoplasmic reticulum, elements of the Golgi complex, and zymogen granules, undergo mitosis in a manner similar to that seen in the developing pancreas. Cells in the parenchyma of the tumor grow as disarrayed cords and sheets, are randomly oriented with respect to each other, and do not form acinar structures. However, when in contact with the adventitial surface of blood vessels, the tumor cells palisade and form a polarized layer of cells with their zymogen granule-rich poles oriented away from the vessel lumen. Only in this area of the tumor is a basal lamina present that underlies the basal plasmalemma of the reoriented epithelial cells. Freeze-fracture electron microscopy of tumor cells in the parenchyma shows extensive disruption of tight junctions whose sealing strands are randomly distributed over the entire plasmalemma. Gap junctions are infrequent and when present are often enclosed by tight-junctional strands. Intramembrane particles are randomly distributed over the cell surface. Both the absence of basal lamina and derangement of the junctional complexes may account in part for the altered morphogenesis of this tumor.     

Accumulation of DNA damage and cell death after fractionated irradiation

The purpose of this work was to determine how fractionated radiation used in the treatment of tumors affects the ability of cancer as well as normal cells to repair induced DNA double-strand breaks (DSBs) and how cells that have lost this ability die. Lymphocytic leukemia cells (MOLT4) were used as an experimental model, and the results were compared to those for normal cell types. The results show that cancer and normal cells were mostly unable to repair all DSBs before the next radiation dose induced new DNA damage. Accumulation of DSBs was observed in normal human fibroblasts and healthy lymphocytes irradiated in vitro after the second radiation dose. The lymphocytic leukemia cells irradiated with 4 × 1 Gy and a single dose of 4 Gy had very similar survival; however, there was a big difference between human fibroblasts irradiated with 4 × 1.5 Gy and a single dose of 6 Gy. These results suggest that exponentially growing lymphocytic leukemia cells, similar to rapidly proliferating tumors, are not very sensitive to fraction size, in contrast to the more slowly growing fibroblasts and most late-responding (radiation therapy dose-limiting) normal tissues, which have a low proliferation index.