Supernatant derived from a human hepatocellular carcinoma cell line (PLC/PRF/5) activates a population of T-suppressor cells

Harvest fluid derived from a primary hepatocellular carcinoma cell line (PLC/PRF/5) inhibited the incorporation of 3H-thymidine into PHA-activated human lymphocytes. A similar effect was observed when lymphocytes were pre-incubated with the tumour supernatant and washed prior to mitogen activation. Not only did the tumour supernatant inhibit 3H-thymidine incorporation by mitogen-activated lymphocytes, but it also inhibited production of the lymphokine leucocyte inhibitory factor (LIF). In experiments designed to establish whether a component of the tumour harvest fluid was activating a population of suppressor cells, normal mononuclear (MN) cells were treated with the PLC/PRF/5 or embryonic fibroblast supernatant for 48 h, after which they were washed and added to normal mitogen-activated lymphocyte cultures. Only cells pretreated with the PLC/PRF/5 supernatant suppressed mitogenesis. The cell responsible for the suppressor effect was a T cell, which after a further 24 h in culture liberated a suppressor factor responsible for inhibiting lymphocyte function. Although the nature of the factor/s in the PLC/PRF/5 supernatant responsible for activation of the T-suppressor cell population is unknown, it is suggested that this mechanism may be important in protecting the tumour from the immune response.     

The labelling index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry

Bromodeoxyuridine (BrdUrd) incorporation and flow cytometry were used to measure human tumour kinetic parameters in vitro and in vivo. The technique was validated by comparison of labelling index estimates of mouse tumours in vivo and in vitro using BrdUrd and flow cytometry with tritiated thymidine (3HdThd) autoradiography. Similar labelling indices were obtained with both in vivo and in vitro incorporation into DNA of the two different precursors. Measurements of human tumour labelling indices were similar following in vitro incubation with either BrdUrd or 3HdThd. The use of BrdUrd allowed the visualization of a population of S-phase cells that did not appear to incorporate BrdUrd or 3HdThd. The human tumour labelling indices obtained with BrdUrd incorporation were similar to previously reported values using autoradiography studies. Preliminary studies demonstrated that significant human tumour labelling could be achieved with an intravenous injection of 500 mg BrdUrd.     

Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin

We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the total cell population incorporated significant levels of 35S into DNA. This result presumably reflected the fact that approximately 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (less than 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent Km for all 4 dNTPs was 50-100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [3H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.     

Inhibition of human tumour cell proliferation by analogues of adenosine

The effects of adenosine and several structural analogues of adenosine upon thymidine incorporation into human tumour cells and rat cervical lymphocytes were investigated. The analogue NECA, which has equal specificity for the A₁ and A₂ receptor, had the most inhibitory effect on lymphocyte proliferation while the A₁ agonists had limited effects, suggesting that these cells possess principally A₂ adenosine receptors. In the case of human tumour cells, however, the most inhibitory effect on proliferation was obtained with the A₁-specific analogues. The general order of inhibitory effects of adenosine analogues on thymidine incorporation in human tumour cells was: S-ENBA>CPA=R-PIA>S-PIA>NECA. These findings suggest that in the cells presently studied the A₁ adenosine receptor predominates. Removal of exogenous adenosine by growth in the presence of adenosine deaminase inhibited thymidine incorporation. The effect of adenosine removal lends further support to the proposal that adenosine has some, as yet unidentified, regulatory role in the control of human tumour cell proliferation. © 1997 John Wiley & Sons, Ltd.     

Specificity of growth inhibition of melanoma by 4-hydroxyanisole

An experimental study using human melanoma (NEL-MI), rat hepatoma (Fu5-5), and human kidney (293-31) cell lines was undertaken in order to evaluate the antitumor activity of 4-hydroxyanisole (4-OHA) in vitro. Prior reports have indicated highly specific antitumor activity of 4-OHA against melanoma cells in vitro. This specific antitumor activity has been proposed to be due to the oxidation of 4-OHA by tyrosinase to cytotoxic oxidation products. Dose-dependent cytotoxicity was observed when cells were cultured for 72 h in the presence of 4-OHA. At 100 microM, 4-OHA produced growth inhibition of 62%, 32%, and 55% in melanoma, hepatoma, and kidney cell lines, respectively. No effect was seen at 10 microM 4-OHA. 1,000 microM 4-OHA produced 100% kill. Tyrosinase activity was detected only in melanoma cells. The effect of 100 microM 4-OHA on the incorporation of 3H DNA precursors in melanoma, hepatoma, and kidney cells was also studied. Thymidine incorporation was inhibited in all three cell lines at the lowest cell density tested, with the greatest inhibition seen on melanoma cells. As cell density increased, the effect of 4-OHA on thymidine incorporation decreased. With respect to RNA synthesis, 4-OHA significantly reduced the incorporation of uridine in all three cell lines, with the greatest effect in melanoma cells. Cell density also affected the inhibition of uridine incorporation, but to a lesser extent than that observed on thymidine incorporation. The effect of 4-OHA on leucine incorporation was modest and uninfluenced by cell density. Thus, cytotoxicity of 4-OHA may involve two different mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transforming growth factor-beta-induced mitogenesis of human bone cancer cells

We report for the first time the bifunctional effects of transforming growth factor-beta on the growth of cloned human osteosarcoma cells (Htb96). Cell growth was assessed by determining the cell number, replication index and [3H]-thymidine incorporation following 48 hours incubation of cultured Htb96 cells with the peptide. Exposure of cells to concentrations of TGF-beta upto 40 pM caused a mitogenic response, concentrations between 40 and 800 pM failed to stimulate cell growth and higher doses caused an inhibition of cell proliferation. The initial cell density was found to alter the responsiveness of Htb96 cells to TGF-beta; stimulation of proliferation was less profound at high and low cell densities. The observed cell density- and growth factor concentration-dependent effects of TGF-beta on the growth of tumour cells might suggest the existence of an autocrine regulatory mechanism. Furthermore, by demonstrating a sensitivity to inhibition by indomethacin, we conclude that the proliferative effect of TGF-beta is at least, in part, dependent on the de novo synthesis of prostaglandins.     

Brain tumour stem cells: the undercurrents of human brain cancer and their relationship to neural stem cells

Conceptual and technical advances in neural stem cell biology are being applied to the study of human brain tumours. These studies suggest that human brain tumours are organized as a hierarchy and are maintained by a small number of tumour cells that have stem cell properties. Most of the bulk population of human brain tumours comprise cells that have lost the ability to initiate and maintain tumour growth. Although the cell of origin for human brain tumours is uncertain, recent evidence points towards the brain's known proliferative zones. The identification of brain tumour stem cells has important implications for understanding brain tumour biology and these cells may be critical cellular targets for curative therapy.     

Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.     

Estimating the selective advantage of mutant p53 tumour cells to repeated rounds of hypoxia

The tumour suppressor gene, p53, plays an important role in tumour development. Under low levels of oxygen (hypoxia), cells expressing wild-type p53 undergo programmed cell death (apoptosis), whereas cells expressing mutations in the p53 gene may survive and express angiogenic growth factors that stimulate tumour vascularization. Given that cells expressing mutations in the p53 gene have been observed in many forms of human tumour, it is important to understand how both wild-type and mutant cells react to hypoxic conditions. In this paper a mathematical model is presented to investigate the effects of alternating periods of hypoxia and normoxia (normal oxygen levels) on a population of wild-type and mutant p53 tumour cells. The model consists of three coupled ordinary differential equations that describe the densities of the two cell types and the oxygen concentration and, as such, may describe the growth of avascular tumours in vitro and/or in vivo. Numerical and analytical techniques are used to determine how changes in the system parameters influence the time at which mutant cells become dominant within the population. A feedback mechanism, which switches off the oxygen supply when the total cell density exceeds a threshold value, is introduced into the model to investigate the impact that vessel collapse (and the associated hypoxia) has on the time at which the mutant cells become dominant within vascular tumours growing in vivo. Using the model we can predict the time it takes for a subpopulation of mutant p53 tumour cells to become the dominant population within either an avascular tumour or a localized region of a vascular tumour. Based on independent experimental results, our model suggests that the mutant population becomes dominant more quickly in vivo than in vitro (12 days vs 17 days).     

Fucolipid metabolism as a function of cell population density in normal and murine sarcoma virus-transformed rat cells.

The incorporation of isotopically labeled fucose into the lipids of normal and murine sarcoma virus-transformed rat cells as a function of cell population density was examined. When normal cells were seeded at low cell density, the levels of the major fucolipids, i.e., fucolipids III and IV, were substantially reduced, but then they increased as the cells approached confluency. This variation in synthesis of fucolipids III and IV appeared to be primarily related to cell density and not to cell growth. Chase experiments revealed that the reduced level of fucolipids III and IV in sparse normal cells is due to decreased synthesis rather than to increased catabolism. In contrast to the observations with normal rat cells, the high level of fucolipid III and the low level of fucolipid IV in murine sarcoma virus-transformed rat cells was shown to be independent of cell population density.     

Fucolipid metabolism as a function of cell population density in normal and murine sarcoma virus-transformed rat cells

The incorporation of isotopically labeled fucose into the lipids of normal and murine sarcoma virus-transformed rat cells as a function of cell population density was examined. When normal cells were seeded at low cell density, the levels of the major fucolipids, i.e., fucolipids III and IV, were substantially reduced, but then they increased as the cells approached confluency. This variation in synthesis of fucolipids III and IV appeared to be primarily related to cell density and not to cell growth. Chase experiments revealed that the reduced level of fucolipids III and IV in sparse normal cells is due to decreased synthesis rather than to increased catabolism. In contrast to the observations with normal rat cells, the high level of fucolipid III and the low level of fucolipid IV in murine sarcoma virus-transformed rat cells was shown to be independent of cell population density.     

Uridine transport and RNA synthesis in growing and in density-inhibited animal cells

Both chick embryo fibroblasts and mouse 3T3 cells reduce the rate at which they incorporate H³ uridine into RNA as their growth becomes inhibited at high cell density. This reduction occurs as a function of the cell population density, and with chick embryo cells (in contrast to 3T3 cells) it is not accompanied by significant medium alterations. This indicates the importance of the cell population density in the control of cellular metabolism. The decline in H³ uridine incorporation is paralleled by a decline in the rate of uptake of the isotope into the acid-soluble pool, suggesting that decreased entry of H³ uridine into the cell, rather than a decreased rate of RNA synthesis, is responsible for the reduced rate of incorporation into RNA of density-inhibited cells. This suggestion was confirmed by finding that when the restriction on uridine uptake was overcome by increasing the concentration of uridine in the medium, the density-dependent inhibition of uridine incorporation was largely reversed. We conclude that, even though the rate of H³ uridine incorporation into RNA is reduced three- to five-fold in density-inhibited cells, the rate of synthesis of pulse-labeled RNA continues at 70 to 85% of the rapidly-growing rate.     

Reversible inhibition by human serum lipoproteins of cell proliferation

Normal human serum or plasma was studied for the presence of inhibitors of cell proliferation by assaying inhibition of incorporation of labeled thymidine into acid-insoluble fraction using human FL cells. Lipoprotein fraction obtained by gel filtration through Sepharose 4B and by KBr density gradient centrifugation was found to play a major part of the inhibitory activity of the serum. It was also shown that the inhibitory activity resides in low-density lipoprotein (LDL). The addition of the lipoprotein fraction to growing FL cells caused an early decrease in the transport of uridine and thymidine across the membrane. This change in the permeability of membrane was followed by the preferential inhibition of DNA synthesis and a reduction in the percentage of mitotic cells in the cell population. The inhibition of the growth was reversible and was observed in various types of cells irrespective of species.     

Modification of cell cholesterol content of rat tumour cells

Summary Among the different constituents of the cell membrane, lipids have been poorly studied with respect to their role in the immunogenicity of tumour cells and their influence on the expression on tumour-associated antigens. Since liposome-associated antigens are more potent immunogens when the lipid matrix is in a rigid state, we have modified the lipid composition of rat hepatoma cells by incorporation of cholesteryl hemisuccinate (CH) into the lipid matrix, and studied its effect on the tumorigenicity and immunogenicity of these tumour cells in syngeneic animals. A slight and significant decrease of tumorigenicity of CH-enriched D23 cells was observed when 2×10³ cells were injected SC, whereas with a higher tumour cell challenge there was no difference in the tumorigenicity of untreated or treated cells. The immunogenicity of CH-treated cells was tested by IP immunization with 10⁷ or 10⁶ cells followed 1 week later by an SC challenge with 2×10⁴ viable D23 cells. No statistical difference was observed between the immunogenicity of CH-enriched cells and that of control cells on either tumour incidence or tumour growth rate. In addition, similar experiments performed with the spontaneous mammary carcinoma SP4 showed that CH-enriched SP4 cells were of lower immunogenicity and unable to induce a significant memory immunity. This lack of effect of the CH treatment on the immunogenicity was not related to the absence of incorporation of CH, since the CH treatment increased the cell lipid rigidity as determined by the increase of fluorescence anisotropy of the diphenyl hexatriene probe. These results obtained in two weak immunogenic tumour models underlined the need for further studies before such a lipid modification of cancer cells is applied in human immunotherapy trials.Attaché de Recherche au CNRS, Fellow of the Royal Society (European Science Exchange Programm) from 1. 4. 1981 to 30. 9. 1981     

Assessment of epidermal growth factor receptor (EGFR, ErbB1) and HER2 (ErbB2) protein expression levels and response to lapatinib (Tykerb, GW572016) in an expanded panel of human normal and tumour cell lines

OBJECTIVE: Lapatinib (Tykerb, GW572016), a potent inhibitor of the catalytic activities of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (ErbB2), inhibits population growth of selected EGFR and HER2 overexpressing cell lines. Previous studies with a small number of cell lines suggest a correlation between overexpression of EGFR and/or HER2 and sensitivity to growth inhibition by lapatinib; however, the precise determinants of lapatinib selectivity for tumour and/or other cells remain unclear. MATERIALS AND METHODS: To clarify the determinants of its selectivity in cultured cells, lapatinib-induced cell population growth inhibition and relative EGFR and HER2 protein expression were quantified in 61 different human tumour cell lines from 12 tumour types, two oncogene transformed human cell lines and two normal human cell cultures. Using statistical tools to analyse the data, a model describing the relationship between lapatinib IC(50) (the response variable) and EGFR and HER2 expression and tissue type (explanatory variables) was derived. CONCLUSION: The results suggest that simultaneous consideration of EGFR and HER2 expression, as well as tissue type yields the best determinant of lapatinib selectivity in cultured cells.     

Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarize the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.     

Actions of exogenous heparan sulfate and hyaluronic acid on growth and thymidine incorporation of normal and transformed human fibroblasts. A comparison with the effects of high cell density and low serum concentration and a warning against thymidine incorporation as a measure of DNA synthesis

Treatment of normal human (WI-38) cells with exogenous heparan sulfate (HS) reduced cell growth and incorporation of radio-isotope-labeled thymidine (TdR) into DNA. In spite that growth of their transformants (WI-38 CT-1) was enhanced by HS treatment, transformed cells also decreased in TdR incorporation thereby. This peculiar observation was explained by a reduction of TdR uptake, leading to a decrease in specific radioactivity of newly synthesized DNA. The changes in cell growth and TdR incorporation by HS treatment were revealed to be similar to the changes with increasing cell density rather than by serum starvation.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

Changes in cellularity induced by radiation in a solid tumour.

The growth, and cellular responses of Morris hepatoma 3924 A to a locally-administered dose of 3750 R X-rays were studied using the following parameters; (1) relative tumour volume changes; (2) tritiated thymidine (3H-TdR) incorporation into DNA; (3) tumour DNA content and (4) cellular analysis, including 3H-TdR labelling index, mitotic index, aberrant mitotic frequency and relative cell density. Before depression of tumour growth, cell proliferation is temporarily interuppted. As proliferation is reinitiated, a short-lived synhcrony and prolongation of cell-cycle traverse are reflected in (a) the labelling index and mitotic index, (b) the relative cell density, and (c) the rate of incorporation of 3H-TdR into DNA. Within 4 days after radiation, cell proliferation and 3H-TdR incorporation are significantly depressed. Simultaneously there are reductions in both the relative cell density and tumour DNA contents, and these remain depressed as the tumours initiate regression. From these studies, it is apparent that the cellular responses to radiation insult occur well in advance of measurable volume changes and are observed both in tumours that continue to regress and in those that initiate regrowth.     

Lesch-Nyhan mutation: the influence of population density on purine phosphoribosyltransferase activities and exogenous purine utilization in monolayer cultures of skin fibroblasts

In extracts of normal and Lesch-Nyhan (LN) heterozygous skin fibroblast monolayer cultures, hypoxanthine-guanine (H-G) and adenine (A) phosphoribosyltransferase (PRT) activities are correlated. These activities vary concomitantly during the life cycle of a culture: Peak activities occur during exponential growth. The ratio of H-GPRT to APRT activity can manifest heterozygosity at the LN locus when H-GPRT activity, per se, is close to and unreliable different from the normal range. If 2 or 8 × 10⁴ cells are planted per 35 mm petri dish, a strain with clearly heterozygous levels of H-GPRT activity in its cell extract may not reveal its genotype by simultaneous measurement of adenine and hypoxanthine incorporation into its acid-insoluble fraction one day after subculture. In contrast, a 1:1 coculture of normal and mutant cells yields the ratio of adenine to hypoxanthine incorporation expected from the behavior of each cell type separately. In heterozygous cultures at the higher population density, the incorporation of hypoxanthine relative to adenine is 28% greater than in those at the lower population density. A 1:1 mixture of normal and mutant cells exhibited a 20% increase in the relative incorporation of hypoxanthine over the same four fold increase in population density. These observations appear to provide a quantitative expression of the phenomenon of metabolic cooperation between contacting H-GPRT-negative and H-GPRT-positive cells.