Br J Cancer74: 999–1004). To define regulation of TN further and investigate its function in ovarian cancer, a range of cell line models were studied. Concentrations of secreted TN in media from cultures of ovarian fibroblast cell lines were at least 100-fold greater than from carcinoma cell lines. Evidence for paracrine regulation of TN secretion was obtained by co-culture of carcinoma cells with fibroblast cells wherein secretion into the media was greater than from fibroblasts alone. Transforming growth factor (TGF)-β1, insulin-like growth factor (IGF)-II and progesterone all stimulated TN secretion while human choriogonadotropin (hCG), follicle-stimulating hormone (FSH) and γ-interferon inhibited secretion. TGF-β1 produced the greatest stimulation of TN in cultured fibroblasts and its co-expression with TN was examined in primary ovarian tumours. There was a significant association between the presence of moderate–strong expression of TN and TGF-β1. Evidence for TN having a functional role in ovarian carcinoma was obtained from adhesion and migration assays. The PE01, PE04, SKOV-3 and 59M cell lines all demonstrated marked adhesion to plastic coated with TN relative to the control protein bovine serum albumin (BSA) and expressed α2β1 and α3β1 integrins. The SKOV-3 cell line migrated more rapidly through TN than through BSA indicating that TN can facilitate migration of ovarian carcinoma cells. © 1999 Cancer Research CampaignThe extracellular matrix glycoprotein tenascin-C (TN) is overexpressed in the stroma of malignant ovarian tumours particularly at the interface between epithelia and stroma leading to suggestions that it may be involved in the process of invasion


Cell fusion has been used to analyse the genetic determinants of metastasis at the cellular level. Highly metastatic mouse melanoma cells were fused with diploid mouse lymphocytes and a range of hybrid clones isolated and tested for tumorigenicity and metastatic potential by s.c. injection into newborn, histocompatible, sublethally-irradiated mice. Although almost all clones tested were tumorigenic, most had considerably reduced metastatic potential. This suggests that tumorigenicity and metastasis are determined by different genetic elements. Histological examination of primary tumours produced by metastatic and non-metastatic hybrid cell lines showed that an essential step in the production of metastases is the separation of tumour cells from the main tumour mass and their movement into the surrounding tissues. The primary tumours of a metastatic hybrid cell line showed local invasiveness whereas those of a non-metastatic cell line did not.


Despite substantial improvements in childhood cancer survival, drug resistance remains problematic for several paediatric tumour types. The urgent need to access novel agents to treat drug-resistant disease should be expedited by pre-clinical evaluation of paediatric tumour models during the early stages of drug development in adult cancer patients.50 values ranged from 1-200 nM and drug potency correlated both with DTD levels and drug-induced apoptosis. However, synergy between RH1 and cisplatin or doxorubicin was only seen in low DTD expressing cell lines. In clonogenic assays RH1 IC50 values ranged from 1.5–7.5 nM and drug potency did not correlate with DTD level. In A673 Ewing's sarcoma and 791T osteosarcoma tumour xenografts in mice RH1 induced apoptosis 24 h after a single bolus injection (0.4 mg/kg) and daily dosing for 5 days delayed tumour growth relative to control.The novel cytotoxic RH1  is activated by the obligate two-electron reductase DT-diaphorase  to a potent DNA interstrand cross-linker. In acute viability assays against neuroblastoma, osteosarcoma, and Ewing′s sarcoma cell lines RH1 ICThe demonstration of RH1 efficacy against paediatric tumour cell lines, which was performed concurrently with the adult Phase 1 Trial, suggests that this agent may have clinical usefulness in childhood cancer. Average 5–year survival rates for childhood cancers have improved dramatically over the past few decades, such that 75% of children with cancer can now expect to be cured . RDES and A673 ESFT cells were kindly provided by Sue Burchill . The osteosarcoma cell lines 791T and U2OS were from the Paterson Institute cell bank. All cell lines were maintained in RPMI 1640 or DMEM F12  supplemented with 10% fetal bovine serum. Lysates of rhabdomyosarcoma cell lines were kindly provided by John Anderson .RH1 was provided by Allos Therapeutics Inc. , and dissolved in DMSO. Other cytotoxic anticancer drugs were purchased from Sigma-Aldrich .2 for 7–10 days until visible colonies (>50 cells) were present in the untreated well. All assays were performed in triplicate. Surviving fraction was calculated as number of colonies in the test condition/number of colonies in the untreated well and plotted logarithmically against drug concentration.Clonogenic assays were performed as described earlier .The sulforhodamine B (SRB) assay  was usedProtein lysates were prepared as described earlier  assay was used to measure functional DTD levels in cell pellets of both the sensitive and resistant cell lines. Diphtheria toxin-diaphorase activity was determined by following the spectrophotometric dicoumarol inhibitable fraction of DCPIP reduction ) and shape of the concentration-effect curve. The ratio of RH1 to the cytotoxic agent of interest was fixed from the IC50 values from the SRB assay. Cells were treated with RH1 and cytotoxic for 1 h. Seven drug concentrations were used covering the concentration-effect curve. Linear correlation coefficients (r) were generated for each curve to determine the applicability of the data to the method of analysis. In all experiments, r was >0.9.Combination Index (CI) methodology was used to evaluate multiple drug-effect interactions with CalcuSyn software  . This us6 A673 cells in 0.2 ml serum-free RPMI tissue culture media were implanted by a single subcutaneous (s.c.) injection into the right flank. For 791T studies, 3 × 106 cells in a 50 : 50 mixture of serum-free RPMI tissue culture media and matrigel to a total volume of 0.2 ml were implanted s.c. in the right flank. Tumours were allowed to grow to 200 mm3, at which point animals were randomly assigned to control or treatment groups. RH1 treatment consisted of intraperitoneal (i.p.) injection of 0.4 mg kg−1 daily for 5 days as described earlier (2 divided by 2) was measured daily. When tumour volume reached 1250 mm3, the mice were killed by a schedule 1 method and tumours were excised and formalin fixed and wax embedded before immunohistochemistry for cleaved caspase 3 . All procedures were carried out in accordance with UKCCCR guidelines 1999 by approved protocols (Home Office Project license no. 40-2804).Balb/c-NUDE mice were used for all experiments. For A673 studies, 5 × 10Individual profile plots of tumour volume against days from randomisation were examined. After taking base 10 logarithms the plots were well approximated with straight lines. Formal fitting was then performed using linear mixed effect models, which incorporated animal-specific random intercepts and slopes. Primary focus lay in the difference in average slope between the RH1 and control groups for each tumour type separately. A pooled estimate and test of this primary parameter was obtained over the two tumour groups using meta-analysis methods given the estimated effect sizes and their standard errors.*2) , which have high level DTD activity, and 791T osteosarcoma cells (5 nM) which showed the highest DTD activity. There was also no correlation between sensitivity to cisplatin or doxorubicin and sensitivity to RH1; U2OS osteosarcoma cells and LA-15S neuroblastoma cells had the same IC50 values to doxorubicin but varied three-fold in their sensitivity to RH1, and LA-15S and LA-155n neuroblastoma cells, with the same IC50 values to RH1 varied three-fold in their sensitivity to cisplatin. Thus, RH1 is a potent inhibitor of clonogenicity in these paediatric tumour cell lines, and potency is independent of DTD expression or activity.The efficacy of RH1 at inhibiting colony formation was assessed across the paediatric tumour cell line panel. RH1 potency was superior to the DNA damaging agent cisplatin, and the topoisomerase inhibitor and DNA intercalating agent doxorubicin, both of which are in widespread clinical use against childhood cancer. RH1 was the most potent of the three agents examined, with 1000-fold greater potency than cisplatin, and 19- to 88-fold greater potency than doxorubicin . Clonoge5–7.5 nM . There w50 values ranging from 1 nM for SH-EP1 neuroblastoma cells, to 200 nM for U2OS osteosarcoma cells  also had the only IC50 values for RH1 above 40 nM. There was no correlation between sensitivity to RH1 and sensitivity to doxorubicin. Although there was no correlation between expression levels or activity of DTD and sensitivity to RH1, there was a correlation within cell line pairs. For example, SH-EP1 neuroblastoma cells, with relatively high DTD activity, had 11-fold greater sensitivity to RH1 than SH-SY5Y neuroblastoma cells, which had no detectable DTD activity. This 10-fold difference in sensitivity to RH1 between the high and low DTD-expressing cell lines was also seen with the LA1-5S/55n neuroblastoma cells (IC50 11 vs 120 nM) and the 791T/U2OS osteosarcoma cells (IC50 26 vs 200 nM). The only cell line pair in which there was not a 10-fold difference in IC50 to RH1 was the Ewing's sarcoma cell lines, A673 and RDES, in these DTD activity was similar (83 vs 57 nM min−1 mg−1). Thus, paediatric tumour cell lines are sensitive to RH1 in an SRB assay of population kinetics, and in this assay, sensitivity to RH1 correlates with sensitivity to cisplatin, and within tumour types, with DTD activity.In keeping with the clonogenic assay results, the comparison of RH1 with cisplatin and doxorubicin in SRB assay of cell population growth showed that RH1 was again the most potent agent, with ICma cells . RH1 wasma cells . In the 50 values of 26 and 200 nM. LA-15S and LA-155n cells also underwent differential induction of apoptosis, again correlating with the differences in their SRB IC50 values. With A673 and RDES Ewing's sarcoma cell lines, there was little difference in their IC50 values by SRB assay, reflected by similar levels of apoptosis between the two cell lines . Thus, the effects of the combination of RH1 with cisplatin or doxorubicin were investigated. The efficacy of fixed ratio combinations of RH1 and either cisplatin or doxorubicin was assessed for all eight cell lines again using the SRB cell population growth assay. Calcusyn software was used to calculate synergy (low combination index (CI) values), additivity , and antagonism . Although strong synergy was seen between RH1 and cisplatin in LA1-55n neuroblastoma cells, at all three concentration levels tested, this was not a constant feature across the panel. In SH-EP1 and LA1-5S neuroblastoma cells RH1 was antagonistic with cisplatin at all three concentration levels , and in trations . In LA1-−1 RH1 was sufficient to induce apoptosis in A673 Ewing's sarcoma grown as s.c. xenografts in nude mice. A single i.p. dose of 0.4 mg kg3 before random assignment of tumour-bearing mice to treatment with i.p. injection of RH1 (0.4 mg kg−1) or DMSO control daily for 5 days. No differences were observed between test and control groups of mice in general health or weight during the course of the experiment, suggesting no general host toxicity of RH1 treatment. Analysis of individual tumour growth rates in a mixed linear effects model allowed the slope of tumour growth to be plotted for each animal (vs vehicle-treated mice across the two tumour types verged on significance (P=0.057)  .in vitro where it is more potent than either of the clinically used conventional DNA damaging agents, cisplatin and doxorubicin. RH1 was developed as a substrate for the two-electron reductase DTD, with the aim of selective activation of the agent in tumour tissue. The absolute requirement for and importance of DTD activity for the cytotoxic function of RH1 is controversial. Within modified isogenic pairs of both breast cancer and colon cancer cell lines, RH1 is clearly more effective against the cells expressing higher levels of DTD (M min−1 mg−1), two of the neuroblastoma cell lines studied had DTD activity below this threshold , yet had clonogenic IC50 values for RH1 of 2.5 nM, identical to that for LA-15S cells with DTD activity of 103 nM min−1 mg−1, well above the suggested saturation point for maximum RH1 activation  are exquisitely sensitive to this agent Earlier studies have shown rapid onset of apoptosis within 6 h of RH1 treatment, increasing up to 48 h after drug removal . Althoug50 values significantly higher than the other lines  and were also relatively resistant to cisplatin. The data suggest that there may be potential benefit in combining RH1 with other cytotoxic agents. However, these in vitro methods are merely suggestive of possible synergy in vivo and the possibility of greater toxicity when combining several DNA damaging agents needs careful consideration.According to the CI, doxorubicin and RH1 were synergistic in several cell lines, including U2OS osteosarcoma cells, and LA155n and SH-EP1 neuroblastoma cells. Synergy between RH1 and cisplatin was only observed in LA155n neuroblastoma cells. Interestingly, U2OS and LA155n cells, in which the greatest synergy between RH1 and doxorubicin was observed, were the two most resistant cell lines to RH1 in the SRB assay, with ICin vitro. This level of apoptosis was not maintained at later time points, suggesting that repeat dosing of RH1, as used in the in vivo experiments, would be required for prolonged effect on tumour growth. RH1 inhibited the growth of both A673 Ewing's sarcoma and 791T osteosarcoma cells grown as xenografts in nude mice. This combination of Ewing's sarcoma and osteosarcoma xenograft data is reflective of the clinical situation where RH1 will be initially evaluated against all relapsed childhood solid tumours.In A673 xenografts, RH1 increased the number of apoptotic cells 24 h after a single dose of RH1, in agreement with the kinetics of apoptosis observed in vitro. RH1 induced apoptosis in paediatric xenografts grown in mice, and slowed the growth of these xenografts. This data supports further investigation of the usefulness of this agent in the clinic.In conclusion, paediatric tumour cell lines reflecting tumour types with poor prognosis were sensitive to the novel bioreductive agent RH1, regardless of their expression of DTD. In selected tumour types, RH1 was synergistic with doxorubicin


The derivation of permanent cell lines from 40 resected oesophageal carcinomas has been attempted. Five long-term lines have been established from three adenocarcinomas, one mixed carcinoma and one squamous carcinoma. Molecular and cellular analyses have been carried out on the lines and clones derived from them. Karyotype analysis indicates genetic variation among the clones. HLA-A, -B and -C is expressed constitutively, but not HLA-DR. ICAM-1-expressing phenotypes may have arisen during adaptation to long-term culture. All lines are capable of response to interferon-gamma (IFN-gamma) and all produce transforming growth factor beta 1 (TGF-beta 1). Two lines are resistant to the inhibitory growth effects of the latter, possibly contributing to malignancy. It is anticipated that these lines, originating from histologically different carcinomas, will provide a valuable, continuous resource for the investigation and treatment of these aggressive tumours.


Substitutions adjacent to the amino group in the 2-phenyl ring with a halogen atom or methyl group enhance potency in sensitive breast lines . Transient biphasic dose responses were induced but rapidly eradicated after specific drug exposure periods. Two human prostate carcinoma cell lines were refractory to the growth-inhibitory properties of 2-(4-aminophenyl)benzothiazoles; IC50 values > 30 microM were obtained. Potency and selectivity were confirmed when compounds were examined in the National Cancer Institute's Developmental Therapeutics screen; the spectrum of activity included specific ovarian, renal, colon as well as breast carcinoma cell lines. Moreover, comparing 6-day and 48-h incubations, the exposure time-dependent nature of the biphasic response was corroborated. Differential perturbation of cell cycle distribution followed treatment of MCF-7 and MDA 468 cells with substituted 2-(4-aminophenyl)benzothiazoles. In MDA 468 populations only, accumulation of events in G2/M phase was observed. Two MCF-7 cell lines were established with acquired resistance to CJM 126 , which exhibit cross-resistance to substituted benzothiazoles, but equal sensitivity to tamoxifen and doxorubicin. Compared with standard anti-tumour agents evaluated in the National Cancer Institute in vitro cell panel, benzothiazoles revealed unique profiles of growth inhibition, suggesting a mode(s) of action shared with no known clinically active class of chemotherapeutic agents.


Intereferon-γ (IFN-γ) has some anti-tumour activity in human ovarian cancer. This cytokine inhibited proliferation in three of four ovarian cancer cell lines in vitro. We then compared the action of IFN-γ in two cell lines, one sensitive and one resistant to its growth inhibitory effects. IFN-γ signalling appeared normal in both cell lines, with stat1 DNA binding activity detectable at 30 min. Continuous exposure to IFN-γ for 2–3 days was necessary for an irreversible effect on cell growth and apoptosis in cells sensitive to growth inhibition. During this time there was an increase in mRNA for the CKI p21, but no alterations in mRNA levels for other members of the CKI family. Maintenance of p21 mRNA required continuous mRNA synthesis. mRNA for the transcription factor IRF-1 was also induced in growth inhibited cells with similar kinetics to those observed for p21. Maximal induction of both p21 and IRF-1 mRNA was observed after 2–3 days IFN-γ exposure as the cells became committed to cell death. There was also a rapid increase in p21 and IRF-1 mRNA in cells resistant to the growth inhibitory effects of IFN-γ, but this increase was not maintained. Thus, continuous interaction with the IFN-γ receptor, together with a sustained induction of p21 and IRF-1, is associated with growth inhibitory and apoptotic effects of IFN-γ in ovarian cancer cells. © 1999 Cancer Research Campaign


Gravimetric and histological studies of lymphoreticular tissues during growth of a syngeneic squamous cell carcinoma in Wistar rats show that the regional lymph node anergy reported previously in this system is associated with replacement of paracortical small lymphocytes by large blastoid cells. The regional node continued to gain weight throughout the period of anergy and showed no atrophy and minimal necrosis. Over the same period the spleen increased in both weight and cytotoxicity.


The expression of hepatocyte growth factor/scatter factor (HGF/SF) was studied in 12 mesothelioma cell lines characterized by either an epithelioid or a fibroblast-like phenotype. Conditioned media from these lines were analysed by bioassay and ELISA, and HGF/SF was detected in three cell lines, all with a fibroblast-like or mixed morphology. None of eight epithelioid cell lines expressed the factor. Thus, for these cell lines, the ability to secrete HGF/SF correlated with the cell phenotype. Following on from these observations, two cell lines, BR and BT, with a fibroblast-like and an epithelioid phenotype, respectively, were further investigated. Both cell lines expressed the Met receptor but only BR secreted HGF/SF. Both cell lines responded to exogenous HGF/SF treatment by a change of morphology but in different ways: BR became more elongated and bipolar, while BT formed more spread-out cell colonies. HGF/SF acted as a paracrine effector on the epithelioid BT cells and stimulated both cell-spreading and proliferation. Interestingly, BT cells spread but did not scatter in response to exogenous HGF/SF. In contrast BR cells showed only some stimulation of cell motility with HGF/SF and no increase in cell proliferation was observed. Because HGF/SF was previously found in the pleural effusion fluids of patients with malignant mesothelioma and in paraffin-embedded tumour tissues, it is concluded that HGF/SF may well stimulate the growth and spread of malignant mesothelioma in vivo by paracrine and/or autocrine mechanisms.


XLand BAK have been determined in a panel of 12 human ovarian carcinoma cell lines encompassing a wide range in sensitivity to cisplatin. Whereas BAX, BCLXLand BAK levels did not correlate with sensitivity, there was a statistically significant inverse correlation  between growth inhibition by cisplatin and BCL-2 levels. In sublines possessing acquired resistance to various platinum-based drugs or across a panel of human ovarian carcinoma xenografts, there was no consistent pattern of BCL-2 expression. Two relatively sensitive lines (A2780 and CH1) have been stably transfected with bcl-2 and bclXLrespectively and two relatively resistant lines (A2780cisR and SKOV-3) stably transfected with bax. Overexpression of BCL-2 in A2780 cells led to resistance to cisplatin compared to the vector control when assayed at 48 h post-drug incubation but a significant increase in sensitivity at 96 h. Relative rates of apoptosis at 48- and 96-h post-cisplatin exposure mirrored the growth inhibition. There was no significant difference in sensitivity of the pair of lines by clonogenic assay. No significant changes in chemosensitivity to a variety of DNA-damaging or tubulin-interactive agents were observed in the remaining transfected lines. Taken together, these results suggest that, in human ovarian carcinoma cells, high BCL-2 levels  confers a trend towards sensitivity not resistance to platinum drugs. © 2000 Cancer Research CampaignThe expression of the BCL-2 family proteins, BCL-2, BAX, BCL


The Con A acceptor glycoproteins of murine and human tumour cell lines revealed by two-dimensional fingerprinting on polyacrylamide gels fall into two main categories: constant glycoproteins expressed by all cell lines and variable glycoproteins which are only expressed by particular tumour cell lines. Since the number of variable glycoproteins on a typical fingerprint is 50, fingerprints from different cell lines are readily distinguishable. However the variable glycoproteins are not expressed idiosyncratically and cell lines derived from similar classes of tumours express similar patterns of the variable glycoproteins. For example, murine fibrosarcomas express patterns which are virtually identical with one another. Characteristic patterns are also expressed by murine macrophage tumour lines, human carcinomas and human B lymphoblastoid cells. Thus, the variable glycoproteins behave as a set of linked markers which are indicators of the type of normal pre-neoplastic precursor cell from which a tumour is derived and appear to be a new type of marker for tumour cell classification. Antibodies to these glycoproteins could prove useful in tumour localisation and diagnosis.


Characteristics of a panel of seven human ovarian tumour cell lines are presented. Positive staining with HMFG2 and ultrastructural identification of desmosomes confirmed the epithelial nature of the cell lines. The lines showed wide variations in ploidy, doubling times and clonogenicity in soft agar. Both vimentin and keratin were equally expressed in five lines, one line showed strong preferential expression of keratin and one line showed preferential expression of vimentin. Karyotypic changes associated with ovarian cancer were identified in all the lines. Four of the seven cell lines showed loss of chromosome material distal to 11p13-15. These cell lines offer considerable potential for research into the biology and genetics of ovarian cancer.


Thymidylate synthase (TS) expression has been characterized for a panel of eight human colorectal carcinoma and five human leukaemia cell lines, to relate differences in intrinsic TS activity, protein and mRNA levels to growth inhibition caused by continuous exposure to THYMITAQ, a specific non-classical antifolate TS inhibitor. Although a 20-fold variation in sensitivity to THYMITAQ was found within the colorectal cell line panel (IC50 0.12-2.7 microM), sensitivity was not related to TS activity, TS protein or TS mRNA levels. For the leukaemic cell lines, only a twofold range in sensitivity to THYMITAQ was observed (IC50 0.87-2.3 microM), and this did not correlate with TS activity, TS protein or TS mRNA levels. Across all of the cell lines, TS activity was linearly related to TS protein levels . However, for both the colorectal and leukaemia cell line panels, no relationship was found between TS mRNA/18S rRNA ratios and either TS activity or TS protein, consistent with the importance of post-transcriptional mechanisms in regulating TS activity. Two of the colorectal cell lines (BE and HCT116) and one of the human leukaemic cell lines (HL60), were intrinsically resistant to THYMITAQ (IC50 > 2 microM) in the absence of TS overexpression, suggesting that, subsequent to TS inhibition, events such as DNA repair and tolerance to apoptotic stimuli are also important determinants of sensitivity to THYMITAQ.


The influence of anaesthetics on the in vivo response of B16 melanoma to melphalan was studied using an in vitro cell-survival assay. Three anaesthetics were used, Saffan (Althesin) Sagatal  and Hypnorm. When Saffan was administered to tumour-bearing animals before melphalan there was a significant increase in tumour-cell kill. This effect was not observed with Sagatal or Hypnorm. Maximum increase in tumour-cell kill was achieved when Saffan was administered about 1 h before melphalan, and was dependent on Saffan dose. Clonogenic tumour-cell repopulation after melphalan was rapid (TD - 1 day) and the rate was similar from 2 levels of cell kill. When Saffan was combined with melphalan the repopulation rate was the same as with melphalan alone, and the increased cell kill was reflected in increased growth delay. The in vitro response of B16 melanoma cells to melphalan was unaltered by pretreatment with, or simultaneous exposure to Saffan. The results suggest that the mechanism of the enhanced cell kill in vivo is probably due to an indirect systemic effect, rather than a direct effect on the tumour cells.


Prokineticins are multifunctional secreted proteins that were originally identified as regulators of intestinal contraction but subsequently shown to affect vascular function, hyperalgesia, spermatogenesis, neuronal survival, circadian rhythm, nociception, feeding behaviour, immune responses, haematopoiesis and the development of the olfactory and gonadotropin-releasing hormone systems. Their role in the reproductive tract is still not fully elucidated, although they are reputed to increase microvascular permeability. Expression of prokineticins and their receptors has been reported in the ovary, uterus, placenta, testis and prostate. Their expression has also been reported in various pathologies of the reproductive tract, and future studies will highlight whether inhibition of prokineticin function in these pathologies would be a useful therapeutic target. Prokineticins (PKs) are factors that have been recently described and are involved in a wide array of functions in different tissues through the activation of their cognate receptors. Here, we first give a general background of the structure and functions of PKs and then focus on the role of PKs in reproduction.et al.et al.The PKs are two recently identified proteins with roles in physiological and pathological conditions. The names PK1 and PK2 were assigned to these proteins by Li Dendroaspis polylepis) and named venom protein A (VPRA) PK1 is the human orthologue of a nontoxic protein isolated from the venom of the black mamba , which is essential for the activity of these proteins. Mutations to this sequence, by insertion of a methionine preceding the N-terminal alanine, substitution of the N-terminal alanine with a methionine The PKs are the cognate ligands for two closely related G-protein-coupled receptors (GPCRs), termed PKR1 and PKR2, which share 85% amino acid identity and exhibit the greatest differences in their N-terminal sequences PKRs have been reported to couple either to Gi PKs were initially reported to be expressed in the gastrointestinal tract, where they were shown directly to stimulate contraction of the ileum longitudinal muscle of guinea pigs PKs are also expressed in steroidogenic tissues, including the testis In vitro studies suggest that PKs promote survival and differentiation of the granulocytic and monocytic lineages. Moreover, the expression of PKR1 and PKR2 in progenitor and mature blood cells A role for PKs has also been suggested in haematopoiesis and in regulation of the immune response in vitro showed PK2-induced migration of subventricular zone-derived neuronal progenitors −/− and PKR2−/− mice showed hypoplasia of the olfactory bulb −/− phenotype is strikingly similar to the Kallmann syndrome (KS) in humans, and recently PK2 and PKR2 mutations were identified in several KS patients PK2 expression has been demonstrated in the central nervous system, and a role in supporting neuronal survival has been suggested PKs are also involved in nociceptive sensitization and regulation of feeding behaviour. Administration of PK2 through several routes decreased the nociceptive threshold to thermal and mechanical pain in rats There are two levels at which PKs can modulate reproductive function. As discussed earlier, PKs have an important role in the development of the GnRH system but they can also function directly in reproductive organs.PKs and their receptors are expressed in the testis and the prostate. In the testis, PK1 is predominantly expressed in testosterone-producing Leydig cells, whereas PK2 is restricted to primary spermatocytes It has been proposed that PKs, through their cognate receptors, function as regulators of proliferation and the formation of fenestrae in the human testis vasculature The expression of PKs and their receptors has been reported in the prostate PKs and their receptors are expressed in the ovary, uterus and in various tissues of pregnancy in vitro suggest that PK1 has a role in the proliferation and survival of endothelial cells of the bovine corpus luteum Studies In the non-pregnant uterus, PK1 is expressed in the glandular epithelium, as well as in the endothelial and stromal cells of the endometrium, predominantly in the functional layer Further support for a role of PKs in reproductive function can be gleaned from PKR2 knockout mice, which show hypoplasia of the reproductive tract PK1, but not PK2, PKR1 or PKR2, expression peaks during the midluteal ‘window of implantation’, with immunolocalization to endometrial glandular epithelium, stromal and endothelial cells, and myometrial vascular endothelium as well as smooth muscle Hyperaemia and endothelial leakage at the implantation site are one of the earliest signs of implantation The expression of PK1 and PKR1 peaks in the trophoblast between 8–10 weeks of gestation, with PK1 being localized to the syncytiotrophoblast, minimal expression in the cytotrophoblast and no expression in the extravillous trophoblast It has been proposed that PK1 and PKR1 have an important role in placentation, owing to their expression peaking during the crucial hypoxic period of placentation (8th–10th week) before establishment of the haemochorial circulation et al.Several studies suggest that PKs have a role in pathological conditions of the reproductive tract. In the male, Samson A direct role for PKs in cancers of the female reproductive tract has not been ascertained. In endometrial and ovarian carcinoma, it has been shown that PK1 expression is either absent or reduced compared with normal tissue Hyperplasia and hypervascularity of the stroma are key features in the development of polycystic ovarian syndrome (PCOS). The search for factors responsible for these features has identified strong expression of VEGF and PK1 in PCOS et al.In preeclampsia, the underlying pathophysiology remains unclear but the resulting placenta is relatively hypoxic, with evidence of chronic inflammatory and vaso-occlusive lesions. A study by Chung The diverse spectrum of established and potential regulatory functions of PKs in the nervous, immune, haematopoietic, vascular, gastrointestinal and reproductive systems suggest that they will be the targets of drug development for numerous pathologies. Within the reproductive system, they are expressed in the testis, prostate, ovary, uterus, placenta and neurones regulating GnRH neuronal development. The last of these is the only established function, and substantial experimental research is needed to complement the observational studies in this emerging arena, to establish functions in reproductive tissues. The development of immunoneutralizing antibodies and small-molecule antagonist and agonist analogues of these complex proteins will greatly enhance the elucidation of their functions, with the promise of establishing therapeutic interventions in reproductive pathologies. Such molecules will also aid in the dissection of the relative contribution of each of the PKs and their receptors to reproductive physiology and pathology. Moreover, future research aimed at the development of targeted and tissue-specific knockout of the genes encoding each of these proteins in mice will help to elucidate their specific role in reproductive function.


A highly significant radiosensitising effect was seen in both cell lines (P<0.001 for MGH-U1 and S40b cell lines). This effect was independent of the concentration of the drug and the duration of exposure prior to treatment with ionising radiation. Cell cycle kinetics of both cell lines was not significantly altered with ZD1839 (0.01 μM) as a single agent. A modest induction of apoptosis was observed with ZD1839 (0.01 μM) as a single agent, but a marked induction was observed with the combination treatment of ZD1839 and ionising radiation. These results suggest a potentially important role for ZD1839 in combination with radiotherapy in the treatment of muscle invasive bladder cancer.The epidermal growth factor receptor (EGFR) is expressed in a wide variety of epithelial tumours including carcinoma of the bladder. Stimulation of the EGFR pathway is blocked by ZD1839 (Iressa), a highly selective EGFR tyrosine kinase inhibitor. Radical radiotherapy is an established organ sparing treatment option for muscle invasive bladder cancer and this study has explored the possibility for the use of ZD1839 as a radiosensitiser in this scenario. The effect of combination treatment with ZD1839 (0.01  Carcinoma of the bladder is the second most common malignancy of the genitourinary tract accounting for 13 600 new cases in the UK in 1999 .α (TGF-α). This results in receptor-associated tyrosine kinase activation, which triggers a cascade of downstream signalling pathways resulting in cell cycle progression, proliferation, angiogenesis and cell survival. A wide variety of human tumours including breast, NSCLC, bladder and SCC of the head and neck have been reported as expressing moderate to high levels of EGFR  is one of a family of four closely related transmembrane glycoprotein receptors consisting of an extracellular ligand binding domain, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor exists as an inactive monomer that undergoes homo- or heterodimerisation after activation by ligands such as epidermal growth factor (EGF) and transforming growth factor-adiation , 1997. IZD1839, a synthetic anilinoquinazoline, is an orally active, highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that inhibits ligand-stimulated EGFR autophosphorylation and signal transduction pathways implicated in the promotion of cancer cell growth and proliferation . TreatmeThe established human bladder cancer cell line MGH-U1 and its radiosensitive mutant clone S40b were cultured in Hams F12 media with 10% foetal calf serum, 1% glutamine and 1% penicillin and streptomycin. Both cell lines were kindly donated by Professor T McMillan  and Dr S Powell . ZD1839 (Iressa) was kindly donated by Astra Zeneca Pharmaceuticals .μM for 24 h. The drug was removed by washing twice with PBS. The cells were then trypsinised, counted using a Coulter Multisizer II Cell Counter® and plated into 60 mm dishes with 5 ml of media. The dishes were incubated for 10 days at 37°C and 5% CO2. The media were aspirated and the plates were stained with 1% crystal violet and surviving colonies with greater than 50 cells per colony were counted.Exponentially growing cells from both cell lines were treated with ZD1839 at final concentrations ranging from 0.01 to 10 μM. The drug was changed every 24 h and a further 10 ml of freshly reconstituted ZD1839 was added. On day 7, the cells were trypsinised and counted using a Coulter Multisizer II Cell Counter®.Exponentially growing cells were treated continuously for 6 days with 10 ml of ZD1839 at final concentrations ranging from 0.01 to 1000 μM for 1–6 days prior to irradiation. The drug was replenished daily. Colonies were stained and counted as before. The clonogenic survival data were fitted to a radiation survival curve using the using the linear quadratic equation and the DRFIT Radiation Survival Fitting Program  in a sealed Caesium-137 gamma-irradiator. The irradiated cells were trypsinised, counted using a Coulter Multisizer II Cell Counter® and plated in 60 mm Petri dishes and incubated as before. The process was repeated for exponentially growing cells treated with ZD1839 at concentrations ranging from 0.01 to 10  Program . DiffereThe expression of EGFR was determined using an EGFR Elisa Analysis Kit . Exponentially growing cells were treated with a detector antibody to EGFR and incubated at 37°C for 4 h. After washing with buffer supplied with the kit, horseradish peroxidase-conjugated streptavidin was added and after 1 h absorbance was measured using a Plate Reader at 450/595 nm.μM) for 24 h. The drug was washed off with media twice. For cell cycle distribution analysis at 0 h following exposure to ZD1839 for 24 h, cells were treated as described below. For cell cycle distribution analysis at 24 and 48 h following exposure to ZD1839, 10 ml of media was added and cells were incubated at 37°C for a further 24 and 48 h from the time of exposure to ZD1839. The cells were trypsinised, centrifuged at 1000 r.p.m. for 5 min, then washed with PBS and centrifuged at 2000 r.p.m. for 5 min. Cell pellets were fixed with 500 μl of ice-cold 70% ethanol and quickly vortexed. The cells were centrifuged at 2000 r.p.m. and the supernatant discarded. Ribonuclease A (50 μl of 100 μl ml−1 solution) was added and incubated for 5 min. PI stain (300 μl of 50 μg ml−1 solution) was added and incubated for 15 min. The specimens were analysed using Flow Cytometry at 620 nm.Cell cycle distribution was analysed at 0, 24 and 48 h after exposure to ZD1839 for 24 h using flow cytometry with propidium iodide (PI) staining. Cells were treated with ZD1839 (0.01–10 μM) for 24 h followed by irradiation (2–10 Gy), or received irradiation alone (2–10 Gy). The cells were trypsinised and centrifuged at 1000 r.p.m. for 5 min. The pellets were washed with 500 μl of cold PBS, centrifuged at 500 g for 5 min and 1 ml of binding buffer was added per 106 cells. A measure of 10 μl of 25 μg ml−1 Annexin V–FITC and PI stain (10 μl of 50 μg ml−1 solution) were added and samples were incubated in the dark for 15 min. The samples were analysed using flow cytometry.Apoptosis was evaluated using Annexin V–FITC conjugates and PI staining. Exponentially growing cells were treated with ZD1839 (0.01 μM). The length of continuous exposure to ZD1839 (1–6 days) at concentrations of 0.01–10 μM also had no significant effect on cell counts or on clonogenic survival after 10 days (six doubling times) (data not shown).‘The clonogenic survival of both cell lines was not significantly affected by exposure to ZD1839 for 24 h, compared to that of the controls, at the concentrations studied  and the radioresistant MGH-U1 cell line (P<0.001). For MGH-U1, the survival fraction at 2 Gy (SF2) was 0.88 and at 10 Gy (SF10) was 0.042. For S40b, the SF2 was 0.66 and the SF10 was 0.0079. The SF2 for the combination exposure to ZD1839 for 24 h preirradiation at a concentration of 0.01 μM was 0.83 and 0.42 for MGH-U1 and S40b, respectively. The SF10 for the combination exposure to ZD1839 for 24 h preirradiation at a concentration of 0.01 μM was 0.012 and 0.00146 for MGH-U1 and S40b respectively, 10 values. Prolonged exposure to ZD1839 for 3 and 6 days preirradiation with the drug being replenished daily did not improve the overall radiosensitising effect of the 24 h exposure regimen. In the MGH-U1 cell line, the SF2 for 3 and 6 days exposure was 0.80 and 0.83 and the SF10 was 0.0089 and 0.0109, respectively. In the S40b cell line, the SF2 for 3 and 6 days exposure was 0.43 and 0.456 and the SF10 was 0.00238 and 0.00165, respectively, μM having no statistically significant difference in both cell lines (P=0.8 and 0.84 in MGH-U1 and S40b), ZD1839 demonstrated a significant radiosensitising effect on both the radiosensitive S40b cell line (−1 of protein) than the radioresistant MGH-U1 cell line (16.7 fm mg−1 of protein).Both cell lines demonstrated a moderate expression of EGFR, with the radiosensitive S40b cell line expressing higher levels (21.5 fm mgμM) revealed no demonstrable alteration in the proportion of cells in different phases of the cell cycle in relation to control cells. There was no significant accumulation of cells in the G0/G1 phase of the cell cycle in the MGH-U1 cell line , or in the S40b cell line . Similarly, there was no significant alteration in the proportion of cells in the S phase of the cell cycle in either cell line at either concentration of ZD1839 (15.2% in the control sample vs 16.2 and 16.6% in the 0.1 and 10 μM samples in the MGH-U1 cell line and 28 vs 27.7 and 21.9% in the 0.1 and 10 μM samples in the S40b cell line), Analysis of cell cycle kinetics at 0, 24 and 48 h postexposure to ZD1839 (0.1 and 10 vs 5.3% in control samples for MGH-U1 cells and 17.8 vs 5.7% in the control sample for S40b cells). Following the combination of ZD1839 and radiation, there was a significant induction of apoptosis in both the radiosensitive and the radioresistant cell lines. In the radiosensitive S40b cell line, 24.3% of cells were undergoing apoptosis compared to control values of 5.7% (P=0.04), while in the MGH-U1 cell line, 20.3% of cells were undergoing apoptosis compared to control values of 5.3% (P=0.04), ZD1839 was found to have a modest proapoptotic effect on both cell lines as a single agent resulting in the induction of apoptosis in the MGH-U1 cell line . A similar effect on the S40b cell line was also noted . Treatment of both cell lines with radiation alone resulted in the induction of apoptosis in both cell lines with a greater proportionate induction in the S40b radiosensitive cell line  compared to the radioresistant MGH-U1 line (16.7 fmol mg−1 of protein), although this does not represent particularly high expression. Previous studies demonstrating an inverse correlation between the IC50 of ZD1839 and EGFR expression are in agreement with our findings  pathway  and this was similar in both cell lines. Treatment with radiation alone resulted in the induction of apoptosis in both cell lines and this was significantly more extensive for the radiosensitive S40b cell line than the parent MGH-U1 line . Combination treatment with ZD1839 and radiation resulted in a marked induction of apoptosis in both cell lines compared to control cells . The mechanism by which ZD1839 promotes apoptosis as a single agent and in combination with ionising radiation is currently under investigation  and the lack of any significant alteration in cell cycle kinetics, there is a definite and pronounced radiosensitising effect in the radioresistant MGH-U1 cell line and an even greater effect in its radiosensitive mutant clone S40b. These results, in conjunction with those of a number of other groups, suggest that ZD1839 has a significant effect on potentiating radiation-induced cell death at doses higher than 2 Gy in a wide variety of cells of varying radiosensitivity. The potentiation of cell death induced in the radioresistant and the radiosensitive cell line is an encouraging feature as many tumours are inherently heterogeneous in their radiosensitivity (The possible mechanisms for the potentiation of radiation-induced cell death by ZD1839 remain to be established and require further investigation. However, our study suggests that despite the relatively high concentrations of ZD1839 needed to achieve cytotoxicity in bladder cancer cell lines (ICsitivity . Radical


A series of monoclonal antibodies was produced by immunizing mice with fresh carcinoma of the colon and with the cell line HT-29. These antibodies could be classified into 3 different groups: (a) those which were HT-29 specific and reacted with no other cell line or fresh colon carcinoma samples; (b) antibodies which reacted with HT-29 and were also reactive with a number of other in vitro cell lines; and (c) antibodies which appeared to be specific for carcinoma of the colon cell lines in that they reacted selectively with colon carcinoma cell lines and not with carcinoma lines derived from other tissues. It was clearly shown by immunoperoxidase staining of both normal and neoplastic cells of the gastrointestinal mucosa, from stomach to colon, that these antibodies were not tumour-specific. Indeed, one antibody (250-30.6) has a remarkable specificity for secretory epithelium of any tissue whether it be found in the gastrointestinal tract, respiratory system or urinary system. We question the existence of tumour-specific antigens detected by monoclonal antisera described thus far, and comment on the remarkable specificity of some of the sera produced, emphasized by the secretory cell-specific antibody described herein.


The prostate cancer cell lines PC3 and LNCaP have been shown to lack expression of the tumour suppressor gene MMAC1/PTEN, in contrast to the immortalized non-tumorigenic epithelial lines PNT1a and PNT2. We have measured the effects of reintroduction of wild type (wt) and mutant MMAC1 genes on to these genetic backgrounds, using gene constructs expressing either wt MMAC1 or various mutants deficient in the dual specificity phosphatase domain of the protein. Over-expression of wild type PTEN protein induced cell shrinkage and rounding, but did not result in increased levels of classical apoptosis. Permanently transfected lines containing the MMAC1 gene could only be obtained from the PNT cells, as PTEN expression resulted in rapid loss of both tumour lines. In contrast, mutation of the phosphatase domain resulted in partial attenuation of the phenotypic effects of MMAC1 after transient transfection, and also allowed the derivation of permanent tumour cell lines containing the mutated MMAC1 gene. The results suggest that re-expression of wt PTEN is incompatible with survival of human prostate cancer cells in vitro, and that the full biological activity of this common tumour suppressor requires functions additional to the established protein and lipid phosphatase activities in epithelial systems. © 2000 Cancer Research Campaign


Human foetal keratinocytes were transfected with a recombinant plasmid (pSV6-1) which contained an origin defective SV40 genome. The resulting transformed cell line had many properties in common with previously described SV40-transformed keratinocytes, including expression of simple epithelial-type keratins. It was non-tumourigenic in nude mice at early passages, forming small benign cysts, however, after approximately 46 in vitro passages, these transformed keratinocytes formed invasive squamous cell carcinomas in athymic nude mice. Several in vitro changes were associated with this acquisition of tumourigenicity (a) an alteration in cellular morphology, (b) development of a cytogenetically marked clone and (c) loss of cell surface fibronectin. The loss of fibronectin was also observed in vivo; cysts formed by SV6-1 Bam/HFK produced human fibronectin whereas tumours did not, although both tumours and cysts were laminin- and keratin-positive. These results indicate that the spontaneous development of secondary events in immortalised human cells may lead to the acquisition of a malignant phenotype.


But the compound has demonstrated undesired agonist-like effect depending on cell, tissue and species studied. Using PR-transfected breast cancer cells MDA-MB-231, this report describes the similarities and differences between progesterone- and RU486-mediated effects on cell growth, cell differentiation and, at the molecular level, on the activation of p44/p42 MAP kinases (MAPK). Like progesterone, RU486 inhibited cells growth by arresting the cells in G0/G1 phase of the cell cycle. In contrast to progesterone that induced cell spreading, RU486 induced a multipolar, stellate morphology. RU486-treated cells showed no increase of stress fibers, nor was there any increase of focal adhesions as progesterone-treated cells did. Furthermore, despite of the fact that both compounds inhibited cell growth, RU486 significantly stimulated the activation of p44/p42 MAP kinases whereas progesterone markedly inhibited the activation. Nonetheless, the effects of RU486 were PR-mediated and RU486 was able to antagonize the effect of progesterone on cell growth and focal adhesion. In conclusion, RU486 can act not only as a progesterone antagonist, a progesterone agonist but also induced morphological and molecular changes that were distinct from progesterone-mediated effects in PR-transfected MDA-MB-231 cells. The non-progesterone-like effect of RU486 may be mediated through a pathway that is different from the progesterone-mediated pathway, or it is the result of a blockade of certain critical step(s) in the progesterone-mediated pathway. In any case, undesired side effects of antiprogestin may create clinical complications. PR-transfected MDA-MB-231 breast cancer cells provide a model for studying the functions of progesterone analogues.© 2001 Cancer Research Campaign


CB30865 was the most potent growth inhibitory agent . CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 , which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4–48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action. © 1999 Cancer Research CampaignThree lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC


Phototherapy in the presence of haematoporphyrin derivative has been shown to have a preferential effect on malignant tumours when compared to normal tissue. This communication presents a comparison of the sensitivity to photochemotherapy in vitro of different cell lines. Asynchronous populations of cells were exposed to light in the presence of haematoporphyrin derivative, and found to be inactivated with a comparable efficiency. The lines were of human, Chinese-hamster or mouse origin and had different abilities to form tumours after heterotransplantation into nude mice or transplantation into syngeneic, immunosuppressed mice. Synchronized cells from 4 of the lines showed a similar variation in sensitivity to light throughout the cell cycle. Cells near the middle of interphase showed the highest sensitivity, whilst cells in early G1 were found to be least sensitive towards treatment with haematoporphyrin derivative and light.


The results of the bioassay showed that these compounds possess potent antitumoractivities against the studied human carcinoma cell lines.O-Cholesteryl-O-phenyl-N-phenylphosphoramidate (


In vitro cloned lines derived from a primary nickel-induced rat rhabdomyosarcoma exhibited diverse levels of susceptibility to spontaneous NK activity. The presence of NK target structures was revealed by competition assays on all cloned cell lines, and the NK susceptibility of the tumour lines varied according to their osmotic fragility. Tumour cell lines derived from metastatic lung nodules presented similar NK susceptibilities to cells originating from the primary tumour. However, cloned cell lines differed in their capacity to form lung colonies after i.v. injection, and in their potential for invading lungs after s.c. primary tumour development. No correlation was found between lung colonization potential and NK resistance. Studies of the correlation between metastatic potential and NK sensitivity revealed that (1) all the NK resistant tumour cells were highly metastatic; (2) NK susceptible tumour cells could be either highly or weakly metastatic. Therefore, highly metastatic tumour cells could be either resistant or susceptible to NK lysis. We conclude that the property of resistance to NK contributes to a high metastatic potential. However, other properties could counterbalance and finally prevail over NK susceptibility thus enabling NK susceptible cell lines to be also highly metastatic.


EO9 [3-hydroxy-5-aziridinyl-1-methyl-2-prop-beta-en- alpha-ol] was selected for clinical trial in Europe because of its preclinical profile but also because of its distinct mechanism of bioactivation. Several studies have shown that cells rich in DT-diaphorase may be particularly sensitive to EO9. The present study examined the relationship between DT-diaphorase activity and sensitivity to EO9 in a panel of cell lines largely derived from human and rodent leukaemias/lymphoma and solid tumours. A possible relationship between chemosensitivity and enzyme activity was demonstrated (correlation coefficient 0.796). A number of the human cell lines were established as xenografts in nude mice but, with the exception of HT29, DT-diaphorase specific activity was greatly reduced compared with the corresponding cell lines. These data suggest that in vitro studies of bioactivation of drugs by specific enzymes is unlikely to be relevant for the same tumour in vivo. Except for HCLO, all xenografts failed to respond to EO9 as a single dose. HT29 tumours in vivo had similar DT-diaphorase activity  to the cell line (337) but failed to respond to a single dose or daily dose schedule. A preliminary attempt to investigate an hourly dose schedule demonstrated a modest anti-tumour effect accompanied by enhanced toxicity. Attempts to optimise EO9 exposure parameters to potentiate activity in tumours with high DT-diaphorase activity are under way, but as yet the relevance of this particular enzyme for in vivo EO9 activity requires further investigation.


The activity of diamine oxidase  which converts putrescine into gamma-aminobutyraldehyde in the degradative pathway of polyamine, was studied in 4 human melanoma cell lines, 2 of which produce tumours in greater than 80% of nude mice , whereas the other 2 induce tumours in less than 25% . The activity of DO in these cells varies with the growth rate: 24 h after seeding there is an initial increase in DO activity, followed by a steep decline during exponential growth. At 96 h, when cells reach saturation density, the activity of DO is significantly greater in the highly tumorigenic cell lines than in the poorly tumorigenic cell lines. Kinetic studies show that for the highly tumorigenic lines apparent Km values are 10.6 X 10(-6)M +/- 0.2 (M3Dau) and 14.2 X 10(-6) M +/- 0.6 (M4Beu), whereas for the poorly tumorigenic lines the values are 4.5 X 10(-6) M +/- 0.3. After transplantation into nude mice, the M1Dor cell line, which exhibits a low Km (app.) for DO of which had high Km (app.) value. Km (app.) determination of DO could be an approach for characterizing human melanoma cells differing in their tumorigenic potential in nude mice.


Drug resistance is a major obstacle to successful chemotherapy of primary liver cancer, which is associated with high expression of the multidrug resistance (MDR) gene product P-glycoprotein (Pgp), a multidrug efflux transporter. The most effective single agents in treatment of primary liver carcinoma belong to the anthracycline family, yet several anthracyclines are known to be substrates for Pgp. In the present study, we compared four anthracyclines with respect to cell growth inhibition, intracellular accumulation and cellular efflux using the HB8065/R human hepatoma cell line which is rich in Pgp, and the Pgp-poor parental line HB8065/S. The anthracyclines were also administered in conjunction with the Pgp-modifying agents verapamil and SDZ PSC 833 to assess modulation of resistance. The HB8065/R cells were sensitive to aclarubicin (ACL) and highly resistant to epirubicin (EPI), doxorubicin (DOX) and daunorubicin (DNR). SDZ PSC 833 enhanced accumulation, decreased efflux and increased cytotoxicity of EPI, DOX and DNR in the HB8065/R cells, but none of these effects was seen with ACL. In conclusion, ACL is apparently not transported by Pgp and retains its activity in a multidrug-resistant human hepatoma cell line; such properties can be exploited for clinical purposes.


The cytolethal response to treatment with prednisolone was investigated in vitro in eight human lymphoblastoid cell lines containing varying concentrations of specific cytoplasmic glucocorticoid receptors. A similar response was observed in seven of the lines irrespective of their concentration of cytoplasmic receptors and pharmacological doses of steroid, well above those required to saturate receptors in cell-free extracts, were required for a massive lethal response. One cell line derived from Burkitt's lymphoma was refractory to lethal effects even with pharmacological doses of steroid. A similar unresponsiveness to the cytolethal effect of prednisolone in vitro was observed in fresh lymphoblasts derived from patients with acute lymphoblastic leukaemia despite evidence of a satisfactory clinical response to therapy which included steroid. The resistance of human lymphoblastoid cells to treatment with glucocorticoids in vitro may result from a defect in activation subsequent to the binding of steroid to cytoplasmic receptors.


Sublines of the human small-cell lung carcinoma (SCLC) cell line GLC4 with acquired resistance to teniposide, amsacrine and mitoxantrone  were derived to study the contribution of DNA topoisomerase II alpha and -beta  to resistance for TopoII-targeting drugs. The cell lines did not overexpress P-glycoprotein or the multidrug resistance-associated protein but were cross-resistant to other TopoII drugs. GLC4/VM20x showed a major decrease in TopoII alpha protein  without reduction in TopoII beta protein; GLC4/AM3x showed only a major decrease in TopoII beta protein (to 18%) and not in TopoII alpha. In GLC4/MIT60x, the TopoII alpha and -beta protein levels were both decreased . The decrease in TopoII alpha protein in GLC4/VM20x and GLC4/MIT60x, was mediated by decreased TopoII alpha mRNA levels. Loss of TopoII alpha gene copies contributed to the mRNA decrease in these cell lines. Only in the GLC4/MIT60x cell line was an accumulation defect observed for the drug against which the cell line was made resistant. In conclusion, TopoII alpha and -beta levels were decreased differentially in the resistant cell lines, suggesting that resistance to these drugs may be mediated by a decrease in a specific isozyme.


Four human and 4 rat astrocytomata and mammalian adult brain and spinal cord were examined by indirect immunofluorescence with human serum containing smooth muscle antibody. Cryostat sections of astrocytomata showed staining of the tumour cell cytoplasm and processes while in normal adult brain and spinal cord the entire astrocyte stained. Impression film and tissue culture monolayers of astrocytomata showed staining of cell processes and a fine, filamentous network in the cell body. The reaction with astrocytoma tumour cells was stronger than that with the corresponding normal astrocytes. Specificity of the staining reaction was established by its prevention on neutralization absorptions of the serum with extracts or homogeneous of smooth muscle. The presence of smooth muscle-associated antigen in astrocytes and astrocytomata is indicative of contractile protein providing a mechanism of cell movement in vivo.


FAK; ‘FAK’) is a tyrosine kinase that is localised to cellular focal adhesions and is associated with a number of other proteins, such as integrin adhesion receptors. We performed an immunohistochemical analysis of FAK protein expression to determine the relationship between FAK overexpression and clinicopathological factors in oesophageal squamous cell carcinoma (ESCC). We examined tissue specimens that had been removed from 91 patients with thoracic oesophageal cancer who had undergone surgery between 1983 and 2001. Immunohistochemical staining was performed by the standard streptavidin–biotin method. Seven human ESCC cell lines–TE-1, TE-2, TE-8, TE-13, TE-15, TT, and TTn–and one immortalized human keratinocyte cell line–HaCaT–were used in Western blot analysis. Immunostaining of FAK was seen in the cytoplasm of cancer cells, particularly in cells located in the invasive fronts of cancer nests. FAK overexpression was detected in 54 of the 91 patients (59.3%). Significant correlations were observed between FAK overexpression and cell differentiation (P=0.0057), depth of tumour invasion (P=0.0023), presence of regional lymph node metastasis (P=0.0097), number of lymph node metastases (P=0.0026), and disease stage (P=0.012). The survival rates of patients with FAK-overexpressing cancer were significantly lower than those of patients without FAK-overexpression cancer (P=0.006). The 5-year survival rate of patients without FAK overexpression was 69%, whereas that of patients with FAK overexpression was 38%. On Western blot analysis, FAK was expressed at a high level in TE-1, TE-8, TE-15, and TT cells, at a moderate level in TE-2 and TTn cells, and at a low level in TE13 and HaCaT cells. FAK phosphorylation at tyrosine 397 was demonstrated in proportion to the intensity of FAK in all cell lines except TE15 and HaCaT. In conclusion, FAK overexpression of ESCC was related to cell differentiation, tumour invasiveness, and lymph node metastasis. Consequently, patients with ESCC who had FAK overexpression had a poor prognosis.Focal adhesion kinase (p125 FAK, hereafter referred to as FAK), is a tyrosine kinase that is localised to cellular focal adhesions and is associated with a number of other proteins, such as the integrin adhesion receptors . Tumour stages were classified according to the 5th edition of the TNM classification of the International Union against Cancer (UICC). The evaluation of tumour differentiation was based on histological criteria of the guidelines of the Japanese Society for Esophageal Diseases . The meaSpecimens were fixed in 10% formaldehyde solution and embedded in paraffin. We examined sections that contained both a tumour-invasive portion and normal oesophageal epithelium.−1 penicillin and 100 μg ml−1 streptomycin). TT and TTn were cultured in 1 : 1 Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 medium (Sigma) containing 10% foetal bovine serum and antibiotics, as described above. HaCaT was cultured in DMEM medium (Sigma) containing 10% foetal bovine serum and antibiotics, as described above. All cell lines were cultured to 60−80% confluence.Seven human oesophageal cancer cell lines — TE-1, TE-2, TE-8, TE-13, TE-15, TT, and TTn — and one spontaneously immortalised human keratinocyte cell line — HaCaT — were used. The TE cell lines were kindly provided by Dr T Nishihira  (397]), ; Mab specific for Ki-67 (MIB-1) ; Mab specific for β-actin, Sigma.Antibodies were purchased from the following manufacturers: monoclonal antibody (Mab) specific for FAK (clone 4.47), ; rabbit polyclonal antibody specific for FAK-phosphorylated at tyrosine 397  at 120°C for 5 min for anti-FAK Mab and were autoclaved in 10 mM citrate buffer (pH 6.0) at 120°C for 5 min for anti-Ki-67 FAK Mab , then cooled to 30°C. After incubation with normal rabbit serum for 30 min, the tissue sections were removed by blotting. The sections were then incubated at 4°C overnight with anti-FAK Mab at a dilution of 1 : 1000 in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, then washed in PBS and incubated with secondary antibody for 30 min at room temperature. Immunohistochemistry was performed using a Histofine SAB-PO(M) kit . The chromogen was 3,3′-diaminobenzidine tetrahydrochloride, applied as a 0.02% solution containing 0.0055% H2O2 in 50 mM ammonium acetate–citric acid buffer (pH 6.0). The sections were lightly counterstained with hematoxylin. Negative controls were prepared by substituting normal mouse serum for primary antibody, and no detectable staining was evident.Immunohistochemical staining was performed by the standard streptavidin–biotin (SAB) method. Briefly, each 4-When >40% of carcinoma cells in a given specimen were stained more intensely than the normal epithelium in the same section, the sample was classified as FAKoverexpression (FAK(+)). Ki-67 labelling index was calculated as the percentage of nuclear staining of cells at the invasive front of the tumour in three consecutive high-powered fields (× 400); each field corresponded to a total number of cells ranging from 300 to 1000. We counted at least 1000 cells per sample.M Tris-HCl, pH 7.6, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 1% aprotinin, 1 mM phenylmethylsulphonyl fluoride, 1 mM sodium vanadate). The protein concentration was determined using a BCA Protein Assay Kit . Protein (30 μg) from each cell line was resuspended in sodium dodecyl sulphate (SDS) sample buffer  containing 1 mM dithiothreitol, boiled for 5 min, and subjected to a 5−10% gradient Ready-Gel . Proteins were electrotransferred to a Hybond-enhanced chemiluminescence nitrocellulose membrane . Proteins were immunoblotted by using anti-FAK . The bands were detected using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). For reblotting; membranes were stripped according to the manufacturer's protocol. Proteins were reblotted using anti-FAK[pY397] and anti-β-actin (Sigma). Anti-β-actin (Sigma) antibody served as the control.Lysates from exponentially growing cell lines were prepared in buffer , depth of tumour invasion (P=0.0023), presence of regional lymph node metastasis (P=0.0097), number of lymph node metastases (P=0.0026), and disease stage (P=0.012). However, there was no significant association with age, sex, tumour location, or presence of distant metastasis.FAK expression in ESCC was investigated by immunohistochemical analysis of formalin-fixed, paraffin-embedded specimens using a FAK-specific Mab. In normal oesophageal tissue, immunostaining of FAK was detected in the cytoplasm of the basal cells, parabasal cells, vascular endothelial cells, and leukocytes .It was reported that integrin signalling through FAK leads to the regulation of cell proliferation and survival . Ki-67 iP=0.006; Figure 2The survival rates of patients with FAK-overexpression (+) cancer were significantly lower than those of patients with FAK-overexpression (−) cancer  patients. A number of reports have indicated that FAK may be up-regulated in human tumour cells of diverse origin (We investigated the levels of expression of FAK in seven ESCC cell lines and one immortalised human keratinocyte cell line. All ESCC cell lines expressed various levels of FAK and, except for TE-13, the levels were higher than those in the immortalised human keratinocyte cell line. Although immunohistochemical analysis showed a significant correlation between FAK overexpression and poor tumour differentiation, there was no relationship between FAK expression and cell line differentiation. This discrepancy may have been due to differences between the characteristics of the cell lines and the pathological diagnoses of the primary lesions. There was no relationship between FAK overexpression and p53 status in the ESCC cell lines. fak gene dosage is increased in a variety of cell lines derived from SCC of the head and neck, lung, breast, and colon cancer (25 As our study material was ESCC, the observed overexpression of FAK might have been caused by fak gene amplification.It has been reported that the In our study, patients with FAK overexpression had a poor prognosis for overall survival. However, multivariate statistical analysis showed that FAK overexpression was not a prognostic factor by itself. Therefore, our result was presumably influenced by the factors of cell differentiation, tumour invasion, and lymph node metastasis. Some reports have demonstrated that the degree of cell differentiation is a useful prognostic factor in ESCC patients . As FAK Detection of FAK expression in formaldehyde-fixed paraffin-embedded tissue sections by immunohistochemical techniques is easy, low cost, and quantifiable, and reveals the localisation of FAK overexpression. Intense FAK expression in preoperative biopsy specimens may be an indicator of advanced disease with a high probability of tumour spread. FAK may be a good therapeutic target, the manipulation of which may prevent ESCC cells from invading other organs and spreading into the lymphatic drainage.In conclusion, FAK overexpression is related to cell differentiation, tumour invasiveness, and lymph node metastasis. Consequently, patients whose tumours overexpress FAK have a poorer prognosis than those whose tumours do not.


The acquisition of drug-resistant tumour cells is the main problem in the medical treatment of a range of malignant diseases. In recent years, three new classes of anti-cancer agents, each with a novel mechanism of action, have been brought forward to clinical trials. These are the topoisomerase I (topo I) poisons topotecan and irinotecan, which are both camptothecin derivatives, the taxane tubulin stabilizers taxol and taxotere and, finally, the antimetabolite gemcitabin, which is active in solid tumours. The process of optimizing their use in a combination with established agents is very complex, with numerous possible drug and schedule regimens. We describe here how a broad panel of drug-resistant small-cell lung cancer (SCLC) cell lines can be used as a model of tumour heterogeneity to aid in the selection of non-cross-resistant regimens. We have selected low-fold (3-10x) drug-resistant sublines from a classic (NCI-H69) and a variant (OC-NYH) SCLC cell line. The resistant cell lines include two sublines with different phenotypes towards alkylating agents (H69/BCNU and NYH/CIS), two sublines with different phenotypes against topo I poisons (NYH/CAM and NYH/TPT) and three multidrug resistant (MDR) sublines  with combinations of mdr1 and MRP overexpression as well as topoisomerase II (topo II) down-regulation or mutation. Sensitivity to 20 established and new agents was measured in a standardized clonogenic assay. Resistance was highly drug specific. Thus, none of the cell lines was resistant to all drugs. In fact, all resistant cell lines exhibited patterns of collateral sensitivity to various different classes of drugs. The most intriguing pattern was collateral sensitivity to gemcitabin in two cell lines and to ara-C in five drug-resistant cell lines, i.e. in all lines except the lines resistant to topo I poisons. Next, all sensitivity patterns in the nine cell lines were compared by correlation analysis. A high correlation coefficient (CC) for a given pair of compounds indicates a similar pattern in response in the set of cell lines. Such data corroborate the view that there is cross-resistance among the drugs. A numerically low coefficient indicates that the two drugs are acting in different ways, suggesting a lack of cross-resistance between the drugs, and a negative correlation coefficient implies that two drugs exhibit collateral sensitivity. The most negative CCs (%) to the new drug leads were: taxotere-carmustine (BCNU) (-75), taxol-cisplatin (-58), ara-C-taxol (-25), gemcitabin-doxorubicin (-32), camptotecin-VM26 (-41) and topotecan-VP16 (-17). The most negative correlations to the clinically important agent VP-16 were: cisplatin (-70); BCNU (-68); camptothecin (-38); bleomycin (-33), gemcitabin (-32); ara-C (-21); topotecan (-17); melphalan (-3); and to the other main drug in SCLC treatment cisplatin were: doxorubicin (-70); VP-16 (-70); VM-26 (-69); mAMSA (-64); taxotere (-58); taxol (-58). Taxol and taxotere were highly correlated (cross-resistant) to VP-16 (0.76 and 0.81 respectively) and inversely correlated to cisplatin (both -0.58). Similarly, camptothecin and topotecan were correlated to cisplatin but inversely correlated to VP-16 and other topo II poisons. From the sensitivity data, we conclude that collateral sensitivity and lack of cross-resistance favours a cisplatin-taxane or topo I-topo II poison combination, whereas patterns of cross-resistance suggest that epipodophyllotoxin-taxane or topo I poison-cisplatin combinations may be disadvantageous.


The overall level of chromatin compaction is an important mechanism of radiosensitivity, and modification of DNA methylation and histone deacetylation may increase radiosensitivity by altering chromatin compaction. In this study, we investigated the effect of a demethylating agent, a histone deacetylase(HDAC) inhibitor, and the two agents combined on radiosensitivity in human colon and breast cancer cell lines.In this study, we used RKO colorectal cancer cell line and MCF-7 breast cancer cell lines and normal colon cell lines. On each of the cell lines, we used three different agents: the HDAC inhibitor sodium butyrate(SB), the demethylating agent 5-Aza-2'-deoxycytidine(5-aza-DC), and radiation. We then estimated the percentage of the cell survival using the XTT method and experimented to determine if there was an augmentation in the therapeutic effect by using different combinations of the two or three of the treatment methods.After treatment of each cell lines with 5-aza-DC, SB and 6 grays of radiation, we observed that the survival fraction was lower after the treatment with 5-aza-DC or SB than with radiation alone in RKO and MCF-7 cell lines(p < 0.001). The survival fraction was lowest when the two agents, 5-aza-DC and SB were combined with radiation in both RKO and MCF-cell lines.In conclusion, 5-aza-DC and SB can enhance radiosensitivity in both MCF-7 and RKO cell lines. The combination effect of a demethylating agent and an HDAC inhibitor is more effective than that of single agent treatment in both breast and colon cancer cell lines. Epigenetics is an important intracellular procedure that can change the genetic information of the cells that is transmitted during cell division without changing the sequences of the DNA bases . Of the DNA methylation is carried out by DNMT (DNA methyltransferase), usually when a methyl group is added to the cytosine residue of a CpG island, which is a group of repeated CpG sequences . AberranHistone alteration is another epigenetic mechanism of regulating transcription. The histone octamer consists of a core, which is encircled by double stranded DNA to form a nucleosome. Two enzymes are associated with histone deacetylation – histone acetyltransferase and histone deacetylase(HDAC) . HDAC taThe biggest difference between the mechanisms of epigenetics and genetics is that epigenetics can be reversed by using certain chemical substances . Also, tRecently, there has been growing interests in the substances that regulate cellular radiosensitivity as a strategy to increase tumor radiosensitivity. There are reports that HDAC inhibitors and demethylating agents enhance radiosensitivity ,12-14. HHuman colon cancer cell lines RKO , breast cancer cell line MCF-7 , and normal colon cell line DDC-112 CoN (ATCC) were used. RKO and MCF-7 cell lines were cultivated in Dulbecco's modified Eagle's medium (DMEM)/F12  combined with 10% fetal bovine serum and 1% penicillin/streptomycin using a humidified cultivator that maintained 37°C and 5% CO2. The normal cell line was cultivated using the same cultivator in Dulbecco's modified Eagle's medium (DMEM) combined with 10% fatal bovine serum.After melting 5-Aza-2'-deoxycytidine  in phosphate-buffered saline, and sodium butyrate(Fluka) in sterilized distilled water, they were stored at 20°C and used when needed.6 cells from each cell line were cultured for 24 hours in 100 mm culture dishes, they were divided into three groups. Each group was irradiated with 4 Gy, 6 Gy, or 4 Gy plus additional day of 4 Gy and cultured for 24 or 48 hours after irradiation. The medium used was Dulbecco's modified Eagle's medium (DMEM)/F12(Gibco) combined with 10% fetal bovine serum and 1% penicillin/streptomycin.After 1 × 10After being treated with 5-Aza-2'-deoxycytidin and sodium butyrate, and after having received radiation for the proper dose and duration, the DNA was extracted using a QIAamp DNA Mini Kit . The procedure of bisulfate modification of genomic DNA was performed as follows.After denaturing 2 ug of DNA into 2 M NaOH, the DNA was incubated in 30 ul of 10 mM hydroquinone and 520 ul of 3 M sodium bisulfate (Sigma) for 16 hours at 50°C. Modified DNA was filtered with a Wizard DNA clean-up system  and then denatured again to 3 M NaOH. 3 M NaOH was precipitated in 100% ethanol and 2.5 M ammonium acetate and, then melted in 20 ul of distilled water. AccuPrime SuperMix I  was used for PCR; Modified genomic DNA 1 ul was amplified. The product was confirmed with 2.5% agarose gel. PCR conditions and primers are given in Tables 3 cells each DDC-112 CoN, RKO, and MCF7 in a 96-well plate, 5-Aza-2'-deoxycytidin 4 uM, sodium butyrate 1 mM, and a combination of both were added and then cultivated for 48 hours. An assay was done using a cell proliferation kit II(XTT).After 24 hours of seeding of 3 × 10For comparison of the treatment effect of radiation, the data were converted to a log scale. Then, using SPSS ver. 13.0, the results were compared with ANOVA, and p values less than 0.005 were considered significant. The average and standard deviation were not converted to log scale in the table of statistics; original data's average and standard deviation were documented.We irradiated the RKO cell line with the different dose of radiation and cultured the cells for 24 hours, 48 hours and 72 hours. Then we analyzed the cell survival Fig . For theIn the RKO cell line, all of the tumor suppressor genes were methylated. Half were methylated in the MCF-7 cell line; MINT 1, MINT 31, p16 were methylated and MINT 2, p14, E-cadherin were unmethylated. None were methylated in the CCD-112 CoN cell lines .In the RKO cell line, 56.5% of the cells survived after radiation alone, 47% survived with the addition of 5-aza-DC, and a similar percentage (46%) survived with the addition of SB. The combination of the two resulted in a 39.6% survival rate, showing the synergic effect of the agents (p < 0.001).There was no statistical significance among survival rates after treatment with radiation, 5-aza-DC, and SB in CCD-112 CoN cell lines Table , Fig 4.With the development of molecular radiobiology, recent researches has focused on the molecules and processes that influence the response of cells to radiation. Many different kinds of molecules are known to increase radiosensitivity by influencing the procedures of cell cycle check points, DNA repair, gene transcription, and apoptosis. Recently, studies of epigenetic procedures such as histone deacetylation and DNA methylation have been proposed for enhancing the radiosensitivity of tumor cells.Out of the many demethylating agents and HDAC inhibitors, we chose 5-aza-DC as the demethylating agent and SB as the HDAC inhibitor for our study. 5-aza-DC is a similar molecule to cytidine. Through a covalent bond to DNMT, it decreases the rate of methylation, thus controlling genetic expression. SB is a short-chain fatty acid that targets the activated region of zinc of HDAC. It has a very short half-life .Histone plays an important role in post-translational modification carried out by histone acetyltransferase and HDAC. Oncogenesis is related to inactivation of histone acetyltransferase, and it is thought that hyperactivation of HDAC suppresses the transcription of tumor suppressor genes, therefore playing an important part in carcinogenesis . HypoaceThere have been many hypotheses proposed for how HDAC inhibitors enhances radiosensitivity. First, the chromatic compaction has an important role in radiosensitivity, and according to the degree of compaction, chromatin can be divided into euchromatin and heterochromatin. Euchromatin is at a relaxed state in which genes are actively undergoing transcription. Heterochromatin contains inactivated genes, which, is at a highly organized state. Genes with ongoing active transcription are generally more sensitive to radiation, while when chromatin condenses into a highly organic structure where transcription is inactive, DNA becomes protected from double strand breaks(DSB) and resistant to the effect of radiation. Euchromatin contains histones, which are acetylated and phosphorylated, while heterochromatin contains deacetylated and methylated histones ,20,21. HHypermethylation of DNA is found commonly in tumor cells, and it suppresses the function of genes that participate in tumor suppression or control the cell cycle, apoptosis or DNA repair -4. RecenIn RKO cell lines, the effect of SB was similar to that of 5-aza-DC, while in MCF-7 cell lines, SB was more effective compared to 5-aza-DC. The function of HDAC inhibitor is considered to be related with the methylation level of the genes. Cameron et al  reportedThere are reports that HDAC inhibitors and demethylating agents have a synergic effect ,11,25,26There are some reports that 5-aza-DC or SB increase the radiosensitivity in other field than colon or breast cancer. De Schutter et al  reportedIn this experiment, the survival rates of RKO and MCF-7 cell lines after irradiation showed significant differences. One limitation of this experiment is that the found in where effect of 5-aza-DC and SB were not measured under the equal conditions.5-aza-DC and SB enhanced radiosensitivity in MCF-7 and RKO cell lines. In RKO cell lines, which are in a relatively hypermethylated state, the effect of 5-aza-DC was similar to that of SB; in MCF-7 cell lines, the effect of SB was better than that of 5-aza-DC. In both cell lines, the combined effect of a demethylating agents, and an HDAC inhibitor showed better results than the effect of each agent used alone. However, this experiment was performed in vitro, and further investigation in vivo is needed.5-aza-DC: 5-aza-2'-deoxycytidine; DSB: double strands break; HDAC: histone deacetylase inhibitor; SB: sodium butyrate; TSA: Trichostatin A.The authors declare that they have no competing interests.CH designed this study and revised manuscript; HJC analyzed the data and wrote the paper; SYK corrected the manuscript; KHK and WKK Collected data; JIK and STO conducted this experiment and JSK helped to design study model.All authors read and approved the final manuscript.


Ultrafiltrates from spleen inhibited both DNA synthesis and the proliferation of normal lymphocytes stimulated inculture from both mouse and man without apparent cytotoxicity. However, the same doses of this spleen ultrafiltrate will kill up to two-thirds of the leukaemic lymphoblasts from both mouse and man after 24 h incubation. This unique lymphocytotoxic effect could also be demonstrated on fresh primary cultures of leukaemic lymphocytes and was highly effective on slowly growing established cell lines under crowd culture conditions. Furthermore. ultrafiltrated thymus extract did not affect the DNA synthesis rates of the viability of NC-37 lymphoblasts, which have B cell characteristic. Thymus extract was cytotoxic to Molt cells, which have T cell characteristics.


It has been reported that cytokeratin 8 (CK8) is expressed in all non-small-cell lung cancers (NSCLC). We hypothesized that antigenic changes of CK8 may occur in some NSCLC cell lines. To prove this, Western immunoblot analysis using anti-human CK8 monoclonal antibodies as well as immunohistological staining of CK8 were performed in NSCLC cell lines. As a result, CK8 which had a higher molecular weight than recombinant CK8 was demonstrated in two of eight NSCLC cell lines. In addition, this CK8 contained antigenic epitopes of CA19–9. This CK8 with higher molecular weight, may have played a role in the process of invasion or metastasis of NSCLC. © 1999 Cancer Research Campaign


The extent of tumour, cell kill, produced by treating B16 melanomas with vincristine, cyclophosphamide, 5-fluorouracil and gamma-rays, alone and in combination, was determined using an in vitro colony assay. Cell kill by vincristine was revealed as a reduction in the yield of cells obtained by trypsinization, and as a decrease in the colony-forming ability of the extracted cells. The reduction in cell yield was interpreted as evidence of rapid cell lysis. Cyclophosphamide and gamma-rays also reduced both cell yield and surviving fraction, but in this case the small decrease in cell yield was due to an increase in cell volume. FU had no effect on cell yield, but surviving fraction was reduced. Tumour weight was also measured, and used in conjunction with cell yield and surviving fraction data to calculate the fraction of surviving cells per tumour following treatment with the agents. In combination studies, single doses of two different cytotoxic agents were given either simultaneously, or up to 24 h apart in either sequence, and assays were performed 24 h after the second drug was given. Combinations of vincristine + cyclophosphamide and 5-fluorouracil + gamma-rays were chosen because they had been shown by other workers to exhibit marked schedule dependency, including considerabl potentiation, against leukaemic cell lines. However, in the B16 melanoma there was no evidence of schedule-dependent cell killing with either of these combinations. For all sequences studied, the fraction of surviving cells per tumour was slightly greater than the predicted additive response calculated from single-drug controls.