Transitional increase in growth fraction estimated by Ki-67 index after irradiation to human tumor in xenograft

The cell kinetics of human tumor cells (HSG) in vivo were estimated by Ki-67 index to analyze the biological behavior of quiescent cells after irradiation. The increment of Ki-67 index was dose-dependent up to 15 Gy irradiation. The Ki-67 index of tumor cells increased gradually from 35+/-5.0% before irradiation to 56+/-5.0% at day 5 after single irradiation of 15 Gy. Flow-cytometric study showed that the G1 population was decreased and the G2M population was elevated at day 5 after the irradiation. In fractionated irradiation, the time-course in the change of the Ki-67 index was similar to that of single irradiation. It was likely that tumor cells were recruited into the cell cycle from quiescent phase after irradiation. We consider the Ki-67 index to be a reliable indicator of growth fraction and useful to estimate the cell kinetics of irradiated tumors in vivo. The growth fraction estimated by Ki-67 index during radiotherapy is expected to be useful in predicting tumor response to radiation therapy.     

In vitro evaluation of B-CLL cells apoptotic responses to irradiation.

Defective apoptosis is a mechanism which could possibly explain B chronic lymphocytic leukemia (B-CLL) cell accumulation. Differences in evolution and prognosis of B-CLL patients may be due to heterogeneity in apoptotic cell death. We studied the apoptotic response to in vitro gamma radiation of blood mononuclear cells from 18 untreated B-CLL patients. In cells irradiated with 2, 4 or 8 Gy and then cultured for 20 hours, the percentage of trypan blue excluding (viable) cells was not modified (>92%). An apoptotic response to irradiation was detected in the majority of the patients, but the individual percentage of apoptotic cells varied widely (8 to 81% after 8 Gy irradiation) in individual cases. The flow cytometric analysis of nick-end DNA labeling demonstrated a dose effect of irradiation, particularly in patients with an apoptotic response of over 20%. In the future, a valuable clue to the selection of irradiation regimens for B-CLL patients may be the investigation of correlations between in vitro radiation-induced apoptosis and the in vivo response to radiation therapy.     

Hydroxyurea enhances methylnitrosourea skin tumorigenesis when given shortly before, but not after, the carcinogen

To study the relationship between epidermal DNA synthesis andcarcinogenesis, hairless mice of both sexes were given a singletopical application of 1 mg N-methyl-N-nitrosourea (MNU) inacetone. A control group received only MNU, whereas other groupswere injected i.p. with 5 mg hydroxy-urea (HU), 1 h, 45 minand 15 min before, simultaneously with, and 15 min, 30 min and45 min after MNU application. The production of skin tumorswas recorded and the results were assessed with accepted statisticalmethods. Injection of HU shortly before a single applicationof MNU enhanced skin carcinogenesis, and when HU is injected30 min before MNU, the enhancement seems to be most pronounced.HU administered simultaneously with or following MNU application,did not alter the production of tumors. The cell kinetic situationin the epidermis at the time of a carcinogen application, andthe modulation of the cell kinetic reaction to the carcinogenby any type of post- or pretreatment, may influence tumorigenesis.     

Maturation of dendritic cells in the presence of living, apoptotic and necrotic tumour cells derived from squamous cell carcinoma of head and neck

Dendritic cells (DC) have been recently used as vaccines for stimulation of tumour-specific immunity in various types of cancer. Since data about interactions of DC with tumour cells derived from head and neck cancer are not available, in our study we investigated the effects of head and neck squamous cell carcinoma (HNSCC) cell lines on the maturation of DC. We found that immature DC efficiently internalise necrotic cells, but not living and apoptotic tumour cells. Although apoptotic cells induced a partial maturation of DC, they were not able to stimulate the secretion of IL-12. In contrast, necrotic tumour cell preparations from all three HNSCC cell lines induced the mature phenotype and IL-12 production by DC. Moreover, necrotic cells synergistically augmented stimulatory effects of monocyte-conditioned medium on the maturation of DC. Thus, DC-based vaccination utilizing necrotic tumour cells as a source of tumour antigens, even in combination with inflammatory stimulus, seems to be a suitable strategy for adjuvant immunotherapy in HNSCC.     

Changes in cell cycle of human hepatoma xenograft in nude mice after irradiation]

Cell kinetics in human hepatoma xenograft in nude mice after gamma irradiation was studied using flow cytometry (FCM) method and the changes of AFP in the xenograft were measured by radioimmunological assay. After 10 Gy irradiation, a marked tumor growth delay for 10 days was observed. Cell cycle analysis revealed an acute but temporary block of cell cycling at G2. About 58% cells were in the G2 phase lasting for 90 hours post-irradiation. A concomitant decrease in serum AFP determined by RIA was also observed. The results indicate that the human hepatoma was quite radio-sensitive.     

Comparative analysis of necrotic and apoptotic tumor cells as a source of antigen(s) in dendritic cell-based immunization.

There is considerable controversy as to whether necrotic (lysate) or apoptotic tumor cells serve as the superior source of multiple tumor-associated antigens (TAAs) to pulse dendritic cells (DCs) for immunotherapeutic applications. Here, we show that standard procedures to induce apoptosis by UVB irradiation unequivocally result in a mixed population of viable, apoptotic, and necrotic tumor cells, necessitating additional purification. We used highly enriched apoptotic versus lysate of B16 melanoma cells to examine whether or not there are important distinctions between these two sources of TAAs for loading of DCs. Our results demonstrate that although some differences exist between the two forms of TAAs in expression of heat shock proteins, as well as production of interleukin-12 by pulsed DCs, their respective capacities to mature DCs phenotypically, as well as to elicit both effective immune priming and antitumor therapeutic efficacy in vivo when presented by DCs, are equivalent.     

Gemcitabine radiosensitizes multiple myeloma cells to low let, but not high let, irradiation.

The radiosensitizing properties of gemcitabine in relation to low Linear Energy Transfer (LET) particles (Cobalt 60) and high-LET particles (alpha-RIT (213)Bi-radiolabeled CHX-DTPA-B-B4) were analyzed. Three multiple myeloma cell lines (LP1, RPMI 8226, U266) were irradiated with or without 10 nM gemcitabine 24 h prior to radiation. Gemcitabine led to radiosensitization of LP1 and U266 cells with low-LET (Radiation Enhancement Ratio: 1.55 and 1.49, respectively) but did not radiosensitize any cell line when combined with high-LET.     

Increase of angiogenic growth factors after hepatic artery embolization in patients with neuroendocrine tumours

In the event of diffuse hepatic metastases, hepatic artery embolization (HAE) can be a successful treatment option in patients with well-differentiated neuroendocrine tumours (NET). However, embolization causes hypoxia which stimulates angiogenesis and therefore tumour growth. This study investigates angiogenesis activity following HAE by measuring vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and C-terminal proendothelin-1 (proET-1) in blood. Twelve patients with well-differentiated NET and liver metastases underwent HAE. VEGF, ET-1 and proET-1 were measured before embolization and the days following treatment during hospitalization. Mean levels during treatment were compared with those at baseline. From 12 patients, 90 blood samples were obtained before and daily for 8 days following HAE. Mean (±SE) VEGF level at baseline was 116 (±33) ng/l which increased after HAE to 313 (±46) ng/l at day 6, followed by a gradual decrease. ProET-1 showed a similar pattern, with a mean baseline level of 9.2 (±2.0) pmol/l and the highest level of 40.8 (±5.7) pmol/l at day 6. Some fluctuations were observed for ET-1, with maximum levels at day 3 compared to baseline levels. In patients with well-differentiated NET who underwent hepatic arterial embolization, angiogenic growth factors increase temporarily. This implies a need to investigate the effect of anti-angiogenic drugs as an adjuvant therapy to embolization.     

Some effects of products from necrotic regions of tumours on the in vitro migration of cancer and peritoneal exudate cells

The invasion of normal tissues by cancer cells and the infiltration of tumours by macrophages to some degree involves active translatory movements by these cells. As necrosis is a common occurrence in solid tumours, we have studied the interactions of saline extracts from the necrotic regions of rat Walker-256 tumours and mouse Gardner lymphosarcomas on the transmembrane, in vitro migration of Walker and Gardner cancer cells, and rat and mouse peritoneal macrophages. Necrotic extracts enhanced cell migration independently of chemotactic activity, an effect which was partially reduced by Trasylol, an inhibitor of neutral proteases. Rat liver lysosomal preparations which contain lower levels of neutral proteases than necrotic extract, inhibited or had no effects on cell migration, and the lysosomal stabilizer hydrocortisone did not inhibit the action of necrotic extracts. The results indicate that necrosis may enhance the migration of cancer cells out of tumours and the migration of macrophages into them. The extracts act partially through their neutral protease content. In contrast to the enhancement of cell detachment by necrotic extracts, which previous work has shown to be partially mediated by their indirect effect in causing lysosomal labilization and which is partially inhibited by hydrocortisone, this indirect mechanism is not demonstrable in the enhancement of cell migration. The results indicate that the consequences of necrosis are worthy of consideration in attempts at understanding the biology of solid tumours.     

DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells

Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. However, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter region of the CDKN2A tumor suppressor gene, we were able to quantify the fraction of plasma DNA derived from tumor cells. In the plasma samples of 30 unselected cancer patients, we detected quantities of tumor DNA from only 3% to as much as 93% of total circulating DNA. We investigated possible origins of nontumor DNA in the plasma and demonstrate here a contribution of T-cell DNA in a few cases only. To investigate the possibility that plasma DNA originates from apoptotic or necrotic cells, we performed studies with apoptotic (staurosporine) and necrotic (staurosporine plus oligomycin) cells in vitro and with mice after induction of apoptotic (anti-CD95) or necrotic (acetaminophen) liver injury. Increasing amounts of DNA were found to be released in the supernatants of cells and in the blood plasma samples of treated animals. A clear discrimination of apoptotic and necrotic plasma DNA was possible by gel electrophoresis. The same characteristic patterns of DNA fragments could be identified in plasma derived from different cancer patients. The data are consistent with the possibility that apoptotic and necrotic cells are a major source for plasma DNA in cancer patients.     

Differential suppression of the tumorigenicity of HeLa and SiHa cells by adeno-associated virus.

Adeno-associated virus (AAV) is well known for suppression of oncogenesis in rodents, but its inhibitory effects on human carcinoma are less well understood. We report the differential ability of AAV to inhibit the tumorigenicity of two human cervical carcinoma cell lines. The wild-type AAV-2 DNA carried by a pSV2Neo vector was transfected into HeLa cells, which contain 50 copies of human papillomavirus type 18 (HPV-18), and SiHa cells, which contain 1-2 copies of HPV-16. About 1-3 copies of AAV genome were introduced per cell. AAV transfection moderately reduced the growth rate and anchorage-independent activity of the cells. In nude mice, the size of tumours arising from SiHa cells was reduced by 87%, in contrast to no reduction in tumour size arising from HeLa cells. This suggests that the differential suppression exerted by AAV may be due to differences in HPV copy number. To define the region that is responsible for the oncosuppression, mutation analyses were conducted. The results of nude mice assays showed that both the replication gene and inverted terminal repeats of AAV were important for the inhibition. This study may provide a model system for further studies on the underlying mechanism of AAV oncosuppressive activity.     

Participation of Fas-mediated apoptotic pathway in KB, a human head and neck squamous cell carcinoma cell line, after irradiation

In head and neck clinical oncology, recurrent cancer after initial irradiation therapy is no longer sensitive to irradiation. To explore the irradiation resistance in head and neck squamous cell carcinoma, a human cell line, KB, derived from the floor of the oral cavity was used. The participation of the Fas-mediated apoptotic pathway was suggested by the upregulation of the surface Fas molecule, the reduction of the apoptotic cell fraction after inhibition of caspase 8 which is a Fas-related initiator caspase, and the changes in Fas-related genes after irradiation. Therefore, it is suggested that disruption of the Fas-mediated apoptotic pathway participates in the acquisition of irradiation-resistance in HNSCC.     

Expression of c-jun and c-fos in apoptotic cells after DNA damage

Apoptosis was induced in HeLa cells by exposure to 50 µM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for various time intervals (up to 120 min). Apoptotic death was confirmed by the microscopic observation of cell blebbing, cell granulation, and cell aggregation. Cells also showed loss of phospholipid symmetry as judged by immunofluorescent microscopy with fluorescently labeled phosphatidyl serine-specific annexin V. In addition, staining of cells with ethidium bromide showed the presence of genomic DNA apoptotic bodies. The protein expression levels of c-jun and c-fos increased in DNA-damaged HeLa cells after MNNG treatment in a time-dependent fashion. Although the levels of c-fos increased rapidly during the first 30 min and remained high for 2 hr, the increase in c-jun expression was more gradual and slower (60-120 min) after MNNG treatment. These results are consistent with the conclusion that c-fos is important in the initial stages (commitment phase) of apoptosis and c-jun is involved in the late stages (execution phase) of apoptosis induced with alkylating agents     

P-glycoprotein expression does not change the apoptotic pathway induced by curcumin in HL-60 cells

Purpose One of the mechanisms responsible for the multidrug resistance (MDR) phenotype of cancer cells is overexpression of so-called ATP-dependent drug efflux proteins: the 170-kDa P-glycoprotein (P-gp) encoded by the MDR1 gene and the 190-kDa multidrug resistance-associated protein 1 encoded by the MRP1 gene. The purpose of the present study was to verify the hypothesis postulating that P-gp expression, apart from enabling drug efflux, confers on the cells resistance to apoptosis by inhibiting caspase-8 and caspase-3.Materials and methods Human HL-60 cells, either drug-sensitive or with the MDR phenotype caused by overexpression of P-gp (HL-60/Vinc) or MRP1 (HL-60/Adr), were treated with the natural dye curcumin at 50 M or with UVC to induce apoptosis. Symptoms of cell death were assessed by morphological observation after Hoechst staining, DNA fragmentation was measured by flow cytometry and the TUNEL method, and caspase-8 and caspase-3 activation and cytochrome c release from mitochondria were measured by Western blotting.Results Curcumin induced cell death in HL-60 cells, both sensitive and with the MDR phenotype, which could be classified as caspase-3-dependent apoptosis, together with cytochrome c release, activation of caspase-3 and oligonucleosomal DNA fragmentation. No active caspase-8 was detected. Also UVC caused caspase-3 activation in both the sensitive and the MDR HL-60 cells.Conclusions Our findings show that there was no correlation between P-gp expression and resistance to caspase-3-dependent apoptosis induced by curcumin and UVC, at least in HL-60 cells. However, we cannot exclude the possibility of parallel P-gp expression and caspase-3 inhibition in some other cell lines, as cancer cells can acquire many different apoptosis-resistance mechanisms.     

Pegylated liposome-encapsulated doxorubicin and cisplatin in the treatment of head and neck xenograft tumours

Purpose: To evaluate the in vitro and in vivo activity of unencapsulated doxorubicin (DOX) and cisplatin (CDDP) and their pegylated liposome encapsulated counterparts (PLED and PLEC) in a subcutaneous model of human squamous cell cancer of the head and neck. Methods: In vitro cytotoxicity was determined by means of the sulphorhodamine B assay and in vivo activity was assessed in terms of tumour growth delay following single intravenous doses of the various agents. Treatment-related toxicity was evaluated by means of serial weight measurement. Results: The IC₅₀ values for DOX (12.1-fold) and CDDP (21.5-fold) were lower than for their liposome-encapsulated counterparts. When the two unencapsulated agents were compared, the IC₅₀ value for DOX was 16-fold lower than that for CDDP. In the in vivo studies, liposomes containing DTPA (PLEDTPA) exerted no effect on KB xenograft tumours when compared to untreated controls (P > 0.1). PLED was significantly more effective than DOX at doses of 2 mg/kg, 4 mg/kg and 8 mg/kg (P < 0.001 for all comparisons). At the 8 mg/kg dose, 7/13 animals treated with PLED were free of disease at 60 days, compared to 0/12 treated with DOX. PLEC displayed superior activity in comparison to CDDP at the 4 mg/kg dose level (P < 0.001), although at doses of 2 mg/kg and 10 mg/kg this comparison only reached borderline statistical significance (0.1 > P > 0.05). The highest dose level of 20 mg/kg was fatal to all animals in the CDDP group but well-tolerated by the animals in the PLEC group. On the basis of serial weight measurements, both PLED and PLEC were shown to be tolerated better than DOX and CDDP. Conclusion: Both PLED and PLEC were shown to exert significant activity against head and neck xenograft tumours, with PLED showing particular efficacy. Received: 2 November 1999 / Accepted: 14 February 2000     

Optimising the combination dosing strategy of abemaciclib and vemurafenib in BRAF-mutated melanoma xenograft tumours

Background:

Resistance to BRAF inhibition is a major cause of treatment failure for BRAF-mutated metastatic melanoma patients. Abemaciclib, a cyclin-dependent kinase 4 and 6 inhibitor, overcomes this resistance in xenograft tumours and offers a promising drug combination. The present work aims to characterise the quantitative pharmacology of the abemaciclib/vemurafenib combination using a semimechanistic pharmacokinetic/pharmacodynamic modelling approach and to identify an optimum dosing regimen for potential clinical evaluation.

Methods:

A PK/biomarker model was developed to connect abemaciclib/vemurafenib concentrations to changes in MAPK and cell cycle pathway biomarkers in A375 BRAF-mutated melanoma xenografts. Resultant tumour growth inhibition was described by relating (i) MAPK pathway inhibition to apoptosis, (ii) mitotic cell density to tumour growth and, under resistant conditions, (iii) retinoblastoma protein inhibition to cell survival.

Results:

The model successfully described vemurafenib/abemaciclib-mediated changes in MAPK pathway and cell cycle biomarkers. Initial tumour shrinkage by vemurafenib, acquisition of resistance and subsequent abemaciclib-mediated efficacy were successfully captured and externally validated. Model simulations illustrate the benefit of intermittent vemurafenib therapy over continuous treatment, and indicate that continuous abemaciclib in combination with intermittent vemurafenib offers the potential for considerable tumour regression.

Conclusions:

The quantitative pharmacology of the abemaciclib/vemurafenib combination was successfully characterised and an optimised, clinically-relevant dosing strategy was identified.     

Telomerase activity in myeloma cells is closely related to cell cycle status, but not to apoptotic signals induced by interferon-alpha.

Telomeres, G-rich structures at the ends of chromosomes are essential for maintaining chromosomal integrity. Most tumor cells contain telomerase, a ribonucleoprotein that elongates telomeric repeats, and it plays an essential role in indefinite proliferation. To better understand regulatory mechanisms of telomerase, in relationship with apoptosis and the cell cycle, we examined telomerase activity in PCM6, an interleukin-6 (IL-6)-responsive, interferon-alpha (IFN-alpha)-sensitive multiple myeloma cell line, using a PCR-based assay. When PCM6 cells were cultured in serum-free media, the addition of IFN-alpha resulted in apoptosis of the cells, but with no influence on telomerase activity. When IFN-alpha was added to the culture with serum plus rIL-6 after serum deprivation, G1-S transition was inhibited and telomerase activity was lower compare to findings in culture with no IFN-alpha. Dose response experiments of rIL-6 and IFN-alpha, and the measurement of telomerase activity of sorted cells in S-phase using CD71, demonstrated a higher activity of telomerase in the samples which contained a larger proportion of cells in S-phase. These data indicate that regulation of telomerase activity is closely related to cell cycle status, in particular cells in S-phase have an high telomerase activity. While telomeres play an important role in cellular senescence, the regulation of telomerase is independent from apoptotic signals induced by IFN-alpha in myeloma cells.     

Influence of irradiation and chemotherapy on the ovaries of children with abdominal tumours.

The ovaries of children with abdominal tumours were studied in 12 autopsy specimens. Ovaries from 25 children who died in accidents or after a short acute disease served as controls. All ovaries from normal children showed follicle growth, but follicle development was inhibited in 67% of the children with abdominal tumours. The effect of treatment with cytotoxic drugs and/or abdominal irradiation on ovarian morphology was investigated. Normal ovaries were found only in children who had received no chemotherapy or a short course. All patients who had been treated with radiation therapy either alone or in conjunction with chemotherapy had severely damaged ovaries: follicle growth was inhibited in all cases, and the number of small non-growing follicles was markedly reduced in most. It is argued that abdominal irradiation might impair follicle development as well as destroy small follicles.