Relationship between cell kinetics and apoptotic effects in TNF-alpha and IFN-gamma-treated human tumour cell lines.

TNF-alpha is a potent inducer of apoptosis and also affects the transit of cells through the phases of the cell cycle. It is thought that the proliferation signalling pathway is related to the apoptosis pathway, the details of this cross-talk are not yet fully understood. In this report, the effects of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on human tumour cell lines with respect to proliferation and apoptosis are examined. The TNF-alpha-sensitive cell lines Me-180 and MCF-7, the resistant cell line TCC-Sup, and the intermediate line 5637 were used. After a one day treatment, the transit through all phases of the cell cycle slowed down and after 3 days stopped completely, as measured with the BrdU-assay and flow cytometry. During the same time however, the levels of c-Myc and Ki-67 expression and the number of cells becoming apoptotic increased. Combined treatment with TNF-alpha and IFN-gamma augmented both the effects on the cell cycle and on apoptosis in the sensitive lines, and had only a minor additional effect on the resistant cell line as compared to single TNF-alpha treatment. The cells becoming apoptotic detached from the culture flask bottom and floated in the medium. Several apoptosis assays were used to prove that the floating cells were indeed apoptotic. As a subsidiary result of receptor measurement, we observed complexes of TNF-alpha receptors I with TNF-alpha receptors II using the related blocking antibodies and I125-TNF-alpha as ligand. The association of proliferative parameters and apoptosis became obvious by plotting the levels of c-Myc expression versus remaining live cells after apoptotic cells were detached. Our data revealed a good linear correlation indicating that high levels of c-Myc render cells sensitive to apoptosis, independent of the treatment, TNF-alpha alone or TNF-alpha in combination with IFN-gamma. The quantitative linear correlation may point to a threshold mechanism.

Effect of cisapride on acid gastro-oesophageal reflux during treatment with caffeine.

About 50% of preterm infants and neonates receiving methylxanthines for respiratory stimulation will develop a pathological gastro-oesophageal reflux (GOR) pattern. In the face of potential GOR-related complications the effect of a concomitant treatment with a prokinetic agent, such as cisapride, should be evaluated. In this study 32 formerly preterm infants were studied simultaneously by 24-hour oesophageal pH monitoring and cardio-respirogram before the presumed end of caffeine treatment. In 14 of these infants a reflux index (RI; percentage of recording time) of more than 4% could be detected (pH <4). Ten of them were treated orally with cisapride (0. 2 mg/kg t.i.d.). Data of pH monitoring, cardio-respirogram and caffeine serum concentrations were obtained before and 5 days after introducing cisapride. The RI and the frequency of GOR decreased significantly with cisapride. The steady-state serum concentrations of caffeine were not influenced by cisapride and the extent of periodic breathing remained unchanged. In conclusion, cisapride has a positive influence on GOR parameters during caffeine treatment without impairing the oral bioavailability or therapeutic effect of caffeine.

Impact of preirradiation of the tumour bed on cell production rate and cell loss of human FaDu squamous cell carcinoma growing in nude mice.

PURPOSE: To explore whether the tumour bed effect (TBE) in FaDu squamous cell carcinoma growing in nude mice is caused by a reduced tumour cell production rate and/or by increased tumour cell loss. MATERIALS AND METHODS: Human FaDu tumours were studied in NMRI nude mice. The volume doubling time (VDT) between 100 and 400 mm3 was determined for tumours in unirradiated subcutaneous (sc) tissues (group 1), tumours in sc tissues preirradiated with 12.5 Gy (group 2), tumours irradiated in situ with 12.5 Gy (group 3), and tumours from group 3 re-transplanted into unirradiated sc tissues (group 4). Labelling index (LI), potential doubling time (Tpot), relative necrotic area and apoptotic index (AI) were evaluated in tumours from groups 1 and 2. RESULTS: The median VDT were 2.6 days (95% CI 2-4) in group 1 and 7.0 days (4-15) in group 2 (p<0.001). The VDT were not significantly different between groups 2 and 3, and group 1 and 4. In groups 1 and 2, the Tpot values (3.1 +/- 0.6 days (SD) versus 2.9 +/- 0.5 days) and the LI were identical (10 +/- 1.5%). The median relative necrotic area was significantly larger in group 2 (37% [23-42]) compared with group (6% [0.3-27]). The apoptotic index was low (0.2%) and did not differ between groups 1 and 2. CONCLUSIONS: The results indicate that the TBE in FaDu squamous cell carcinoma is not caused by a reduced cell production rate in the viable tumour compartment. Rather, the TBE reflects a decreased viable tumour cell compartment due to increased cell loss. Necrosis appears to be the major component of the tumour bed induced cell loss in FaDu tumours, whereas apoptosis has no impact on the TBE in this model.

Impact of overall treatment time on local control of slow growing human GL squamous cell carcinoma in nude mice treated by fractionated irradiation.

BACKGROUND AND PURPOSE: The impact of overall treatment time of fractionated irradiation on local control of slow growing human GL squamous cell carcinoma (SCC) was determined. MATERIALS AND METHODS: Moderately well differentiated and keratinizing human GL SCC with a volume doubling time of 8 days were transplanted subcutaneously into the right hindleg of NMRI (nu/nu) mice and irradiated with 30 fractions under ambient conditions over 2, 3, 4.5, 6 and 10 weeks. Endpoint of the experiments was local tumor control at day 180 after end of treatment. RESULTS: The tumor control dose 50% (TCD50) increased from 40 to 57 Gy when the treatment time was extended from 2 to 10 weeks. The data can be well described by a linear increase in TCD50 with time. The recovered dose per day (D(r)) was 0.28 Gy (95% confidence interval 0.06; 0.48). The fit to the data was not significantly improved by assuming a biphasic (dog-leg) time course with constant TCD50 values in the initial part of treatment followed by a more rapid increase of TCD50 thereafter. CONCLUSIONS: D(r) in GL SCC was significantly less than the value of 1.0 Gy (0.7; 1.3) previously reported for poorly differentiated, non-keratinizing and fast growing human FaDu SCC (Baumann M, Liertz C, Baisch H, Wiegel T, Lorenzen J, Arps H. Impact of overall treatment time of fractionated irradiation on local control of human FaDu squamous cell carcinoma in nude mice. Radiother. Oncol. 1994:32:137-143), indicating important heterogeneity of the time factor between different tumors of the same histological type.

Degradation of apoptotic cells and fragments in HL-60 suspension cultures after induction of apoptosis by camptothecin and ethanol.

Early indicators of apoptosis in mammalian cells are membrane potential breakdown (loss) in mitochondria (MPLM), chromatin condensation, DNA degradation, and phosphatidylserine exposure (PSE) on the outside plasma membrane. One aim of the present study was to determine the kinetics of these characteristics. These changes were measured by flow cytometry using the following methods: membrane potential of mitochondria was analysed using Mito Tracker Green and Red, PSE was analysed using annexin-V-FITC staining simultaneously with propidium iodide (PI) to detect membrane permeability; chromatin condensation was measured using the acid denaturation Acridine Orange (AO) method; DNA degradation was studied by the sub G1 method and the terminal transferase dUTP nick end-labelling (TUNEL) assay (labelling of strand breaks). HL-60 cells were induced to apoptosis by 3% ethanol and 1.5 microM camptothecin (CAM) and the kinetics of the apoptotic cells were measured. The same kinetics were found for chromatin condensation and DNA degradation indicating that these changes appeared at approximately the same time after induction. The MPLM and PSE kinetics showed a considerably later increase indicating that MPLM occurred downstream of DNA degradation and that plasma membrane changes occurred downstream of MPLM. The main aim of the study was to follow the fate of apoptotic cells after the appearance of the initial characteristics. The lifetime of apoptotic cells was studied by chase experiments. The inducing drug was removed after 4 h treatment and the disappearance of apoptoses recorded. An exponential decay was measured with a half life (T(1/2)) of 17.8 h. As a corollary from these experiments, camptothecin was found to induce apoptosis also in G1 and G2 phase cells, however, it took much longer to occur than in S phase cells. Using labelling of the plasma membrane with a fluorescent cell membrane linker, it was possible to show that the majority of apoptotic bodies as well as condensed apoptotic cells contain DNA and membrane. The degradation of these apoptotic bodies follows similar kinetics as those of the condensed apoptotic cells. The membrane remained considerably stable, there was no further loss in the next 7 days, after the first day when the apoptotic characteristics develop. It is concluded that the apoptosis programme is completed within a day and no further steps follow.

Growth of human prostate carcinomas with and without hormone alpha- dehydrotestosterone in nude mice.

OBJECTIVES: The dependence of human prostate carcinoma growth on hormone was studied in xenotransplants in nude mice. The objective was to determine differences in cell kinetic parameters and volume growth of tumors growing with alpha-dehydrotestosterone (alphaDHT) and without alphaDHT. These differences could be used as arguments pro and contra the adaptation versus the clonal selection hypothesis. METHODS: Human prostate carcinomas were xenotransplanted into nude mice. Growth of tumors was observed in castrated male mice without and with implanted osmotic pumps secreting alphaDHT. In a further series of experiments the alphaDHT tubes were removed when the tumors had reached a volume of 0.3 cm3. Tumor volume was measured to determine tumor doubling time with and without alphaDHT. Detailed cell kinetics were analyzed using the bromodeoxyuridine (BrdUrd) method with flow cytometry. Applying the relative movement (RM) and a simulation analysis to parallel single and multiple BrdUrd labelling experimental data we determined transit times through the phases of cell cycle, potential doubling time Tpot, growth fraction (GF) and cell loss. RESULTS: Five human prostate carcinomas were xenotransplanted into nude mice. Tumor take was only achieved when androgen hormone was present. However, when alphaDHT was removed when the tumors had grown to a volume of 0.3 cm3, they continued to grow at nearly the same Td as those tumors with continued alphaDHT application. The BrdUrd experiments, on the other hand, showed considerable increase of Tc and Tpot upon withdrawal of alphaDHT in 4 out of 5 tumors. The GF and labelling index (LI) were maintained at about the same level as alphaDHT consuming tumors. CONCLUSION: While small transplanted tumor pieces do not grow without alphaDHT, larger tumors grow with the same Td after removal of alphaDHT. The slower proliferation shown by the increased Tc and Tpot is balanced by less cell loss. Since GF and LI were maintained at about the same level, we conclude that in our tumors the majority of cells adapted to hormone independence. There was no evidence for the selection model since the tumors continued to grow at about the same speed after hormone depletion. All cell kinetic parameters showed a considerable inter- and intratumoral heterogeneity. A clinical implication may be that hormone ablation therapy should always be supplemented by some other therapy.

Assessing genetic markers of tumour progression in the context of intratumour heterogeneity.

This is a report from the Kananaskis working group on quantitative methods in tumour heterogeneity. Tumour progression is currently believed to result from genetic instability and consequent acquisition of new genetic properties in some of the tumour cells. Cross-sectional assessment of genetic markers for human tumours requires quantifiable measures of intratumour heterogeneity for each parameter or characteristic observed; the relevance of heterogeneity to tumour progression can best be ascertained by repeated assessment along a tumour progressional time line. This paper outlines experimental and analytic considerations that, with repeated use, should lead to a better understanding of tumour heterogeneity, and hence, to improvements in patient diagnosis and therapy. Four general principles were agreed upon at the Symposium: (1) the concept of heterogeneity requires a quantifiable definition so that it can be assessed repeatably; (2) the quantification of heterogeneity is necessary so that testable hypotheses may be formulated and checked to determine the degree of support from observed data; (3) it is necessary to consider (a) what is being measured, (b) what is currently measurable, and (c) what should be measured; and (4) the proposal of working models is a useful step that will assist our understanding of the origins and significance of heterogeneity in tumours. The properties of these models should then be studied so that hypotheses may be refined and validated.

Heterogeneous cell kinetics in tumors analyzed with a simulation model for bromodeoxyuridine single and multiple labeling.

Bromodeoxyuridine (BrdUrd) labeling of DNA and flow cytometry measurement of bivariate BrdUrd-DNA content distributions yield proportions of cells in the cycle phases. After application of BrdUrd, with time, these proportions change according to the cell kinetic parameters of the investigated cell line or tumor. In a previous study of S-phase transit time using the relative movement method, we obtained better fits with S-duration distributions rather than constant values (Baisch and Otto: Cell Prolif 26:439-448, 1993). Now, we have developed a simulation model using asymmetric phase duration distributions in all phases of the cell cycle to fit the experimental data after single or multiple BrdUrd labeling. The model includes transit of cells from proliferating to quiescent compartments in all phases. The results yield the phase duration distributions, mean and median percentages of quiescent cells in all phases, growth fraction, and potential doubling time. The model was used to fit data of five renal cell carcinomas xenotransplanted into nude mice that were obtained after single and multiple labeling up to 93 hours. The estimated phase duration distributions varied from narrow to extremely asymmetric. In particular, TG2M duration and asymmetry were nearly as large as those of G1 phase in some tumors. The contribution of inter- and intratumoral heterogeneity cannot be separated by the simulation model, but evidence of intratumoral heterogeneity is provided by DNA content distributions at extended time spans after BrdUrd labeling.

Impact of overall treatment time of fractionated irradiation on local control of human FaDu squamous cell carcinoma in nude mice.

A series of experiments were performed to determine the impact of overall treatment time on local control of human FaDu squamous cell carcinoma irradiated with 30 fractions under ambient conditions in nude mice. The TCD50 increased with treatment time between 15 days and 10 weeks from 43 Gy to 102 Gy. The data can be well described by a single linear function. The dose recovered per day is 1.0 Gy. However, the data can also be adequately fitted by two components with an initial delay of about 30 days followed by a steep increase at a rate of 1.5 Gy per day. Assuming that the increase of TCD50 is solely caused by repopulation of clonogenic tumor cells, and that the cellular radiation sensitivity in vitro reflects the radiation sensitivity of FaDu cells in vivo, the doubling time of clonogenic tumor cells during treatment is estimated to be approximately 1.8 days for the one-component model and, after an initial delay, approximately 1.2 days for the two-component model. Both values are shorter than the doubling time of clonogenic cells in untreated FaDu tumors and similar to the potential doubling time determined by flow cytometry after BrdUrd labelling. It is concluded that the dose necessary to control FaDu squamous cell carcinoma increases considerably with increasing time of a fractionated radiation treatment. It appears most likely that this increase is caused by repopulation of clonogenic tumor cells; however, other mechanisms such as an increasing fraction of hypoxic tumor cells can not be ruled out at present.

Intratumoral heterogeneity of S phase transition in solid tumours determined by bromodeoxyuridine labelling and flow cytometry.

Cell kinetics of human renal cell carcinomas xenotransplanted into nu/nu mice were analysed using the bromodeoxyuridine (BrdUrd) labelling method. Tumours were removed 0.5-14 h after injection of the BrdUrd solution. The tumour cells were stained with fluorescein isothiocyanate conjugated anti-BrdUrd antibodies and propidium iodide (DNA content). From the flow cytometry data the relative movement was calculated. Relative movement data of variable intervals after BrdUrd labelling were subjected to a fit procedure using log-normal distributions for S phase transition (T(s)). The log-normal distributions were modified by inflation factors in order to get extremely asymmetric distributions. The best fits to the experimental data were obtained using wide asymmetric T(s) distributions, indicating that progression through S phase in solid human tumours is considerably heterogeneous. This implies that the potential doubling time (T(pot)) is longer than calculated from a single measured relative movement value obtained a few hours after BrdUrd labelling.

Sensitivity of urinary quantitative immunocytology with monoclonal antibody 486 P3/12 in 241 unselected patients with bladder carcinoma.

We investigated the sensitivity of quantitative immunocytology with our monoclonal antibody 486 P3/12 in 241 unselected patients with transitional cell carcinoma. Immunocytology yielded a sensitivity of 91.8%, 89.4% and 92.9% for grade 1, 2 and 3 tumors, respectively. Standard cytology was positive in 59.2%, 63.8% and 84.7%, respectively. Deoxyribonucleic acid flow cytometry, used in the first 69 patients, was positive in only 27.7%, 48.6% and 57.1%, respectively.

Nuclear DNA content in 27 pancreatic endocrine tumours: correlation with malignancy, survival and expression of glycoprotein hormone alpha chain.

Paraffin-embedded tissue from resection specimens of 14 functioning and 13 nonfunctioning pancreatic endocrine tumours (PET) was analysed for nuclear DNA content by image cytometry. Data on follow-up (mean 5.5 years) were available in all patients. DNA histograms with a diploid pattern were found in 13 (48%) tumours, while an aneuploid pattern was seen in the remaining 14 tumours (52%). Six (40%) of the diploid tumours and 9 (60%) of the aneuploid tumours were malignant. Survival was shorter in patients with malignant and aneuploid PET (mean 3.5 years, range 0.5-7) than in those with malignant and diploid PET (mean 5.7 years, range 3-8). Human chorionic gonadotropin-alpha was expressed in 3 of 12 benign PET, with 1 being aneuploid, and 6 of 15 malignant PET, with 4 being aneuploid. We conclude from these results that the ploidy pattern of PET allows no discrimination between benign and malignant tumours but may provide prognostic information on the aggressiveness of malignant PET.

Decreased binding of HIV-1 and vasoactive intestinal peptide following plasma membrane fluidization of CD4+ cells by phenytoin.

Plasma membrane fluidity of intact peripheral blood lymphocytes (PBL) of phenytoin-treated nonepileptic patients and phenytoin-treated CD4+ lymphoid cells H9 and K37 was determined by fluorescence anisotropy measurements. Anisotropy values of the membrane probe 6-(9-anthroyloxy) stearic acid were decreased in all cell types as compared with controls, indicating increased plasma membrane fluidity of phenytoin-treated cells. Specific binding of 125I-labeled vasoactive intestinal peptide (VIP) to its cellular receptor CD4 on PBL was decreased in PBL of phenytoin-treated patients as compared with untreated, healthy subjects. Adsorption of a different ligand to the CD4 receptor on PBL, the human immunodeficiency virus type 1 (HIV-1), was likewise abolished to PBL of phenytoin-treated patients and phenytoin-treated CD4+ H9 and K37 cells, as assessed by indirect immunofluorescence. Subsequent HIV-1 infection of phenytoin-treated H9 and K37 cells was reduced as measured by indirect immunofluorescence and p24 antigen production. These data indicate that CD4 receptor availability for VIP and HIV-1 was reduced in phenytoin-treated cells. Using the DNA-specific dye Hoechst 33258, we examined cell cycle phase distributions of HIV-1 adsorbing and nonadsorbing H9 cells, as separated by flow cytometry. The majority of HIV-1 adsorbing cells were found to be in the G2/M phase, while nonadsorbing cells were mainly in the G0/G1 phase, during which plasma membrane fluidity is supposed to be increased. This study indicates that plasma membrane fluidization by phenytoin may serve to disrupt CD4 receptor function and emphasizes the impact of plasma membrane properties on HIV-1 adsorption and infection.

Antiproliferative and cytotoxic effects of single and combined treatment with tumor necrosis factor alpha and/or alpha interferon on a human renal cell carcinoma xenotransplanted into nu/nu mice: cell kinetic studies.

A human renal cell carcinoma serially transplanted into nude mice was treated with recombinant human tumor necrosis factor alpha (TNF-alpha), recombinant human alpha interferon (IFN-alpha), and a combination of both. All treatments resulted in a significantly reduced tumor growth. The greatest effect was obtained with the combination of TNF-alpha and IFN-alpha. This latter treatment completely eradicated tumors which were smaller than 50 mm3 at the beginning of treatment. Cell kinetic analysis using the bromodeoxyuridine technique and flow cytometry revealed a prolongation of the transition time through S-phase from 7.9 h in the case of control tumors to 10.5 h for tumors treated with IFN-alpha and TNF-alpha. Single treatment with either TNF-alpha or IFN-alpha had only minor effects. The bromodeoxyuridine labeling index was unaffected by IFN-alpha (16.6%; control, 15.2%) but was reduced to 12.1 and 11.7% when tumors were treated with TNF-alpha and IFN-alpha plus TNF-alpha, respectively. The calculated potential doubling times were 2.3 and 2.8 days, respectively, for tumors treated with TNF-alpha or IFN-alpha plus TNF-alpha. When treated with IFN-alpha, the potential doubling time (1.7 days) was similar to that of the control (1.6 days), indicating that the main effect of TNF-alpha was antiproliferative. Conversely, the calculated cell loss factors increased after IFN-alpha and combined treatment but not after TNF-alpha treatment. Combined treatment augmented cytotoxicity, but the cell kinetic characteristics of surviving cells remained similar to those of tumors treated with TNF-alpha alone. Histological analysis showed a distinctly reduced mitotic activity but no coagulative necroses after treatment with TNF-alpha. IFN-alpha and, in particular, IFN-alpha plus TNF-alpha induced cytoplasmic vacuolization, nuclear pyknosis, and cell death, which resulted in tumor regression. These data suggest that, in this particular tumor model, TNF-alpha produces mainly antiproliferative effects, whereas IFN-alpha acts via cytotoxic mechanisms.

Combined treatment of rat rhabdomyosarcoma R1H with mitomycin C and X-rays.

The effects of mitomycin C (MTC) alone and in combination with radiation on R1H rhabdomyosarcomas in WAG/Rij rats were studied. Growth delay was determined from tumor volume growth curves and proportion of tumor and host cells from flow cytometry measurements. Only minor growth delays were obtained with MTC at doses near the toxic level while irradiation applied in three fractions of 10 Gy in three weeks yielded growth delays of 27, 44, and 51 days measured at volumes of 2, 5, and 10 cm3, respectively. The combination of 1 micrograms/kg b.w. MTC and 10 Gy irradiation given in three weekly fractions resulted in 21, 28, and 29 days growth delay for the above tumor volumes. The lower effect of combination as compared to irradiation alone is discussed with the flow cytometry data. These data show that early after start of treatment the proportion of tumor cells remains constant in contrast to radiation alone where the fraction of tumor cells decreases because of influx of granulocytes and macrophages into the tumor. In contrast to the reduced effect on tumors the combination treatment was considerably more toxic for the animals than the single treatments.

DNA ploidy and cell-cycle analysis in pancreatic and ampullary carcinoma: flow cytometric study of formalin-fixed paraffin-embedded tissue.

The cellular DNA content of formalin-fixed, paraffin-embedded specimens from 47 ductal adenocarcinomas of the pancreas and 5 adenocarcinomas of the ampulla of Vater was analysed using flow cytometry. Ploidy and the fraction of cells in the S and G2M phases were determined and correlated with tumour stage and grade as well as patients' survival. Cell populations with aneuploid DNA content were observed in 15% of the tumours. The S + G2M fractions ranged between 1% and 10%. Compared to non-neoplastic tissue of the pancreas the S + G2M fraction was significantly higher in the carcinomas. Cox regression analysis revealed the S + G2M fraction as an independent prognostic factor (p less than 0.05). Ploidy was of no prognostic value for survival, but correlated weakly with tumour stage and tumour grade. All patients without lymph node metastases at time of surgery had diploid tumours. Aneuploidy was restricted to tumours in advanced stages and tended to be more frequent in high-grade tumours.