Enhanced sensitivity of human oral tumours to the flavonol, morin, during cancer progression: involvement of the Akt and stress kinase pathways.

Various naturally occurring flavonoids have been found to be cancer-protective in chemically induced animal cancer models and synthetic flavonoid derivatives are being tested for potential chemotherapeutic usefulness in clinical trials. This report demonstrates that human oral squamous carcinoma cells (SCC) are significantly more sensitive to growth inhibition by the naturally occurring flavonoid, morin (3,5,7,2',4'-pentahydroxyflavone) than normal oral mucosa (NOMC) (SCC IC(50) = 115 microM; NOMC IC(50) = 173 micro M; P for difference = 0.009). Structure/function comparisons indicate that both the 2' and 4' hydroxyl groups in morin are required for its tumour selectivity. Morin causes growth arrest in G(2)/M, without inducing apoptosis, and this is associated with induction of GADD45 and phosphorylation and inactivation of the cell cycle kinase, cdc2. Morin also has pleiotropic effects on kinase signalling pathways, including inhibition of activation of protein kinase B by mitogens (but not extracellular-regulated kinases 1/2) and activation of the stress pathway kinases, Jun N-terminal kinase and p38 kinase. p38 kinase activation is functionally important since inhibition of its activation by the specific inhibitor SB202190 partially prevented cell cycle arrest by morin. However, analysis of dose-response relationships reveals that the enhanced tumour sensitivity to morin may be explained by the fact that activation of AKT is inhibited at lower concentrations of morin in carcinomas than normal oral mucosa, whereas Jun N-terminal kinase, p38 kinase and GADD45 are all induced in parallel with the same dose-response curves in carcinomas and normal oral mucosa.

Enhanced sensitivity of human oral carcinomas to induction of apoptosis by selenium compounds: involvement of mitogen-activated protein kinase and Fas pathways.

Prospective studies and recent intervention trials suggest that the risk of some cancers, including respiratory tract cancers, may be inversely related to selenium (SE) intake, and this is supported by strong experimental evidence with chemical-induced animal cancer models. How this cancer-protective effect is mediated is unclear, but interference with the balance of growth/apoptosis during tumor outgrowth is one plausible hypothesis. In general, there is a correlation between the effectiveness of SE compounds as chemopreventive agents in vivo and their ability to inhibit cell growth and induce apoptosis in vitro. This study has investigated the signal transduction pathways affected by SE compounds in biopsies of normal human oral mucosa cells and human oral squamous carcinoma cells (SCCs), using a primary culture system. Two SE compounds were tested: selenodiglutathione (SDG), the primary metabolite of selenite and the most commonly used cancer-protective SE compound in animal models, and the synthetic SE compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC), one of the most potent chemopreventive pharmacological SE compounds. Three novel findings are reported: (a) SCCs were found to be significantly more sensitive to induction of apo ptosis by SDG than normal human oral mucosa cells, though the differences were marginal with p-XSC; (b) both SE compounds induced the expression of Fas ligand (Fas-L) in oral cells to a degree that correlated with the extent of apoptosis induction; and (c) both SDG and p-XSC induced the stress pathway kinases, Jun NH2-terminal kinase (JNK) and p38 kinase, at concentrations causing apoptosis; p-XSC, and to a lesser extent SDG, also activated extracellular regulated kinases 1&2 (ERKs 1&2) and protein kinase-B or Akt. To test their functional involvement, the effect of inhibiting each of these pathways on induction of apoptosis by SDG and p-XSC was determined in SCCs. Inhibiting the ERKs 1&2 or Akt pathways with specific chemical inhibitors (PD98059 or LY294002, respectively) did not affect the extent of apoptosis induced by SDG or p-XSC (with the exception of LY294002, which actually enhanced the level of induction of apoptosis by SDG). The JNK pathway appeared to be most important for induction of Fas-L and apoptosis because concentrations of SB202190 that inhibited activation of both the JNK and p38 kinase (but not ERKs 1&2) in SCC reduced the extent of induction of Fas-L and apoptosis by SDG and p-XSC, whereas lower concentrations that inhibited activation only of p38 kinase did not. This was confirmed by the fact that exogenous expression of a dominant negative deletion mutant of c-Jun (TAM67) reduced the induction of both apoptosis and Fas-L by SDG.

Selenium and signal transduction: roads to cell death and anti-tumour activity.

Accumulated evidence from prospective studies, intervention trials and studies on animal models of cancer have suggested a strong inverse correlation between selenium intake and cancer incidence. Several putative mechanisms have been suggested to mediate the chemopreventive activities of selenium: of these, the inhibition of cellular proliferation and the induction of apoptosis are particularly attractive. The mitogen activated protein kinase (MAPK) pathways are known to be important regulators of cell death and our recent work has focused on the involvement of these pathways in selenium-induced apoptosis in primary cultures of oral cancers and corresponding normal mucosa derived from biopsy material. Using this system, the oral carcinoma cells were found to have enhanced sensitivity to apoptosis when treated with certain selenium compounds compared to normal oral mucosa. Induction of Fas ligand was associated with selenium-induced apoptosis. Signal transduction studies suggests that selenium induces several changes in the MAPK signalling pathways but functional intervention/inhibitor studies indicate that activation of the JNK pathway seems to be most important.

Molecular mechanisms of cancer prevention by selenium compounds.

Selenium compounds that are chemopreventive in animal models inhibit cell growth and induce apoptosis in vitro, and this could explain how they reduce the outgrowth of tumor cells in vivo. Our recent work has shown that primary cultures of oral carcinoma biopsies are significantly more sensitive than normal oral mucosa cultures to induction of apoptosis by a natural selenium metabolite [selenodiglutathione (SDG)], and this is associated with induction of Fas ligand, a well-known mediator of apoptosis in other contexts, and activation of so-called stress kinase signaling pathways, particularly the Jun NH2-terminal kinase (JNK). Heme oxygenase, another marker of stress responses, is also induced by selenite and SDG. The selective activation of the Fas pathway in carcinomas could be responsible directly for their destruction by apoptosis or target them for attack by immunologic responses. In contrast, although the potent pharmacological selenium chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) also induces Fas ligand, heme oxygenase, and stress kinase pathways, apoptosis/Fas induction is not so strongly JNK-dependent and p-XSC does not show tumor selectivity. These differences in mechanism between SDG and p-XSC may be due to the manner in which they induce redox changes in the cells, since although the effects of SDG and p-XSC are prevented by antioxidants such as glutathione or N-acetylcysteine, hydroxyl radical scavengers such as mannitol or pyrrolidine dithiocarbamate only protect against the effects of p-XSC.

Carbonyl reductase: a novel metastasis-modulating function.

To explore reasons for differences in the malignancy of tumors, we have compared two cell lines derived from a mouse lung adenocarcinoma cell line that differ 10-fold in their capacity to form lung metastases from s.c. primary tumors or after i.v. injection. One mRNA encoding carbonyl reductase was identified at a relatively high abundance in the subline with low metastatic capacity but was not detectable in the highly metastatic subline. Transfection of the former subline with a plasmid construct expressing antisense carbonyl reductase rendered the cells highly metastatic. Conversely, the capacity of the highly metastatic cells to metastasize was markedly reduced after transfection with a construct expressing carbonyl reductase. We also found that human prostate cancers show loss of carbonyl reductase expression compared with normal prostate epithelia. These data suggest that carbonyl reductase has an important function in modifying the metastatic behavior of malignant tumors.

An activating mutation in the kit receptor abolishes the stroma requirement for growth of ELM erythroleukemia cells, but does not prevent their differentiation in response to erythropoietin.

We have previously shown that murine ELM erythroleukemia cells can only be grown in vitro in the presence of a stromal feeder layer, or alternatively stem cell factor (SCF), without which they differentiate. When grown in the presence of SCF, ELM cells can still differentiate in response to erythropoietin (Epo), but growth on stroma prevents this. We previously isolated a stroma-independent ELM variant, ELM-I-1, that is also defective in Epo-induced differentiation. We show here that this variant has an activating mutation in the Kit receptor, converting aspartic acid 814 to histidine. Expression of the mutant receptor in stroma-dependent ELM-D cells causes growth factor-independent proliferation and also gives the cells a selective advantage, in terms of proliferation rate and clonegenicity, compared with ELM-D cells grown in optimal amounts of SCF. Expression of the mutant receptor in ELM-D cells also prevents spontaneous differentiation, but not differentiation induced by Epo. Analysis of mitogenic signaling pathways in these cells shows that the mutant receptor induces constitutive activation of p42/p44 mitogen-activated protein kinases. It also selectively inhibits the expression of p66Shc but not the p46/p52 Shc isoforms (as did treatment of ELM cells with SCF), which is of interest, because p66Shc is known to play an inhibitory role in growth factor signaling.

Both stroma and stem cell factor maintain long-term growth of ELM erythroleukemia cells, but only stroma prevents erythroid differentiation in response to erythropoietin and interleukin-3.

Defining how the stromal requirements of hematopoietic progenitors change during leukemia progression is an important topic that is not well understood at present. The murine ELM erythroleukemia is an interesting model because the erythroid progenitors retain dependence on bone marrow-derived stromal cells for long-term growth in vitro, and they also undergo erythroid differentiation in the presence of erythropoietin (EPO) and interleukin-3 (IL-3). In this report, we have shown using neutralizing antibodies that stem cell factor (SCF), insulin-like growth factor (IGF)-1, and integrin signaling pathways are all involved. We then determined whether ELM cells can be maintained long-term without stroma in various combinations of growth factors produced by stroma cells or growth factors for which ELM cells have receptors. This showed that ELM cells could be maintained with high efficiency in SCF alone; furthermore, the cells remained absolutely SCF-dependent and did not become more tumorigenic than cells maintained on stroma. In contrast, ELM cells underwent clonal extinction when serially cloned in IGF1; any cells that survived long-term growth in IGF-1 were found to be IGF1-independent. One important difference between maintaining ELM cells on stroma and growth in SCF is that stroma reversibly inhibits their differentiation in response to EPO and IL-3, whereas SCF does not.

Inappropriate retinoic acid receptor-beta expression in oral dysplasias: correlation with acquisition of the immortal phenotype.

Retinoids are useful in the treatment of premalignant oral lesions and in preventing the occurrence of second primary cancers after resection of the initial primary oral cancer, but long-term prognosis is still poor, presumably due to malignant cells escaping retinoid control. Previous work has shown that loss of expression of retinoic acid receptor beta is one of the most consistent molecular changes during oral cancer progression in vivo. In this report we demonstrate, using a novel panel of primary cultures of oral lesions, that loss of retinoic acid receptor beta expression at the dysplasia stage occurs during the transition from senescent to immortal phenotype but may occur independently to the loss of CDKN2A/p16 expression.

Chemopreventive and growth inhibitory effects of selenium.

There is very convincing evidence that a high dietary level of selenium substantially reduces the incidence of a wide variety of animal cancers. The human epidemiological evidence is less clear cut, but overall suggests that selenium may be protective: the evidence is strongest in men in relation to gastro-intestinal cancers. There is evidence that dietary selenium compounds reduce the formation of DNA adducts by carcinogens. Selenium compounds also inhibit growth in vitro and induce apoptosis. In general, there is a good correlation between the effectiveness of selenium compounds in chemoprevention and growth inhibition, implying that the mechanisms of growth inhibition and chemoprevention may be similar and that a major factor in the chemopreventive effects of selenium compounds in vivo is their ability to retard outgrowth of pre-malignant cells. Various hypotheses have been advanced as to how selenium compounds might prevent tumour cell growth. One is that they cause apoptosis by inducing oxidative stress. However, we have shown that the most potent selenium compound, selenodiglutathione (SDG), a natural metabolite of selenite, does not induce oxidative stress, at least not in the same way as other oxidants such as H2O2 and diamide. Firstly, a partially selenium-resistant variant cell line does not show increased resistance to H2O2. Moreover, SDG does not induce widespread tyrosine phosphorylation, including MAP and SAP kinases, like other oxidants such as H2O2 and diamide and its effects are not reversed by pretreatment with the tyrosine kinase inhibitor, herbimycin. Our experiments with the selenium-resistant variant suggest that a novel selenium-binding protein may be involved in growth inhibition by selenium.

The role of soluble growth factors in inducing transient growth and clonal extinction of stroma cell dependent erythroblastic leukemia cells.

A coculture system of a murine erythroblastic leukemia cell line (ELM-D) with its supportive stromal cell line (MS-5) was established. Long-term growth of ELM-D cells is strictly stroma cell dependent. Interaction between stem cell factor (SCF) and its receptor, c-kit, was demonstrated to be important for stroma cell-dependent growth by anti c-kit neutralizing monoclonal antibody (mAb) inhibition experiments. Significantly, soluble growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or SCF of MS-5 stromal cells (MS-5 CM) could replace the requirement of stroma cells for a considerable period. However, ELM-D cells maintained in these growth factors underwent clonal extinction after 3-6 weeks unless contact with stroma was re-established. Furthermore, IL-3 or GM-CSF acted in a dominant manner in inducing cell death in the presence of stroma cells. Cells showing clonal extinction undergo programmed cell death and do not differentiate. These altered growth properties of ELM-D cells exposed to soluble growth factors or to stroma cells appear to be analogous to those described for T or B cells primed by antigen presenting cells and then grown in growth factors.

Acute intermittent porphyria caused by defective splicing of porphobilinogen deaminase RNA: a synonymous codon mutation at -22 bp from the 5' splice site causes skipping of exon 3.

Acute intermittent porphyria (AIP) results from mutations in the porphobilinogen deaminase (PBG) gene. Three of 14 randomly selected, unrelated patients with the cross reacting immunological material (CRIM) negative form of AIP were found to have previously undescribed RNA splicing defects. Defective splicing of exons 12 and 13 was caused by a C-->G transversion at position -3 of the 3' splice site of intron 11 and a G-->A transition at the first position of intron 13, respectively. Defective splicing of exon 3 was associated with a synonymous codon mutation (CGC-->CGG, R28R) at position -22 from the 5' splice site. Our findings are consistent with previous reports indicating that about 15% of mutations in the PBG deaminase gene that cause AIP affect RNA splicing and add to the evidence that synonymous intraexonic codon mutations may cause disease.

Clinical features and outcome of patients with thin and ultrathin glomerular membranes.

There is considerable disagreement regarding the natural history of renal disease associated with thin glomerular basement membranes (TGBM). We followed 43 patients (19 male), mean age 41.6 years (range 19-73) for a mean of 88 months (48-140). TGBM was recognized in adults when glomerular basement membrane thickness, measured from multiple sites in electronmicrographs of renal biopsy tissue as the harmonic mean, was < 320 nm. At presentation, 95% had microscopic haematuria, 12% macroscopic haematuria, 14% loin pain, 28% proteinuria, and 14% hypertension. There was no difference in GBM width between the sexes (male 258 nm vs. female 251 nm) but there was a significant negative correlation between age and GBM width (r = -0.53, p < 0.001), with older patients having the thinnest membranes. Twenty six patients had ultrathin GBM (< 270 nm), of whom 54% had 3+ haematuria vs. 12% of the group with BM > 270 nm (p < 0.01). In the ultrathin group, 71% had loss of anionic charge from the GBM, vs. 17% in those with membranes which were thin but > 270 nm (p < 0.05). Proteinuria occurred more frequently in those with GBM > 270 nm, 65% vs. 8% in the ultrathin group (p < 0.01). Thin GBM were associated with a benign prognosis, as after a mean follow-up of 85 months (48-140), there was no significant change in either serum creatinine or mean arterial blood pressure. Patients with ultrathin GBM had greater loss of GBM anionic charge, which might result in both an alteration of flow characteristics within the glomerular capillaries and also increased fragility of the glomerular basement membrane with likelihood of rupture and resultant macroscopic haematuria.

Altered selenium-binding protein levels associated with selenium resistance.

Our previous studies have sought to elucidate the mechanism by which selenium inhibits cell growth since the mechanisms involved may be relevant to the chemopreventive properties of selenium. In a previous report, we described the isolation of a selenium-resistant cell line, B19, from a selenium-sensitive parental cell line, C57. In this report we identify biochemical changes that may be responsible for conferring selenium resistance. Altered selenium uptake and intracellular glutathione concentrations were eliminated as possible modes of resistance since these two parameters were similar between the two cell lines. However, when the selenium-containing protein complements of the two cell lines were compared by labelling with [75Se]selenite, both increases and decreases in a number of selenium-labelling proteins were found in the B19 cells. The most striking differences were the presence of two 72 kDa selenium-labelling proteins in B19 cells which could not be detected in C57 cells. The levels of a number of mRNAs encoding antioxidant or detoxification enzymes were also compared between the two cell lines but only minor changes were found. This work suggests that further analysis of the 72 kDa selenium-labelling proteins may reveal insights into the mechanisms of growth inhibition by selenium.

Tissue-specific regulation of the rabbit 15-lipoxygenase gene in erythroid cells by a transcriptional silencer.

The 15-lipoxygenase (lox) gene is expressed in a tissue-specific manner, predominantly in erythroid cells but also in airway epithelial cells and eosinophils. We demonstrate in this report that the 5' flanking DNA of the 15-lox gene contains sequences which down-regulate its activity in a variety of non-erythroid cell lines but not in two erythroid cell lines. The element has characteristics of a transcriptional 'silencer' since it functions in both orientations. The main activity of the silencer has been mapped to the first 900 bp of 5' flanking DNA, which contains nine binding sites for a nuclear factor present in non-erythroid cells but not in erythroid cells. These binding sites have similar sequences and multiple copies of the binding sites confer tissue-specific down-regulation when attached to a minimal lox promoter fragment. The 5' flanking DNA also contains a cluster of three binding sites for the GATA family of transcription factors.

The selenium metabolite selenodiglutathione induces cell death by a mechanism distinct from H2O2 toxicity.

Our previous studies have implicated the selenium metabolite selenodiglutathione (SDG) in the growth inhibitory effects of selenite in vitro. Other work has suggested that reactive oxygen species, the superoxide anion and hydrogen peroxide, may be implicated in selenite toxicity. In this study the mechanism of growth inhibition by SDG and H2O2 has been compared in a mammary cell line, C57. Both SDG and H2O2 had a rapid effect on C57 cells and markedly reduced cloning efficiency within 1 h. However, the mechanisms involved seem to be different, as judged by the following observations: (i) An SDG-resistant cell line (B19) derived from C57 cells is cross-resistant to selenite, but not H2O2; (ii) SDG reduces the levels of the mRNAs for phospholipid hydroperoxide glutathione peroxidase and cytosolic glutathione peroxidase, whereas H2O2 has no effect; (iii) SDG induces both 560 kb and 50 kb DNA fragments, whereas H2O2 only induces 560 kb DNA fragments. This is of interest, since formation of high molecular weight DNA fragments has been recognized as a characteristic of apoptosis.

The selenium metabolite selenodiglutathione induces p53 and apoptosis: relevance to the chemopreventive effects of selenium?

Selenodiglutathione (SDG), the initial metabolite of selenite, is shown to be a more powerful inhibitor of cell growth in vitro than selenite itself. This has been established both with mouse erythroleukaemia (MEL) cells and an ovarian cell line (A2780) which is known to contain wild-type p53. Other seleno-compounds, such as selenomethyl selenocysteine (SMS) and dimethyl selenoxide (DMS), which are potent chemopreventive agents and are known to be metabolized to methylated selenium derivatives directly rather than via SDG, are also growth inhibitory to both MEL and A2780 cells, although less so than SDG or selenite. However, cells growth-inhibited by DMS are more viable than cells growth-inhibited to the same extent by SDG or selenite, suggesting that the methylated seleno-compounds may inhibit cell growth in a different manner from that of SDG or selenite. Our studies of the mechanism of growth inhibition by SDG, have established two facts. First, SDG induces p53 protein levels in cells that contain wild-type p53 (A2780 cells), suggesting that SDG induces the DNA damage-recognition pathway. Secondly, SDG induces apoptosis in MEL cells, as judged by flow cytometry and formation of nucleosomal DNA ladders. However, since p53 mutations have been found to be targetted events in all MEL cells examined, our evidence suggests that induction of apoptosis by SDG is not absolutely dependent on the p53 response pathway.

Transcriptional up-regulation of the mouse cytosolic glutathione peroxidase gene in erythroid cells is due to a tissue-specific 3' enhancer containing functionally important CACC/GT motifs and binding sites for GATA and Ets transcription factors.

Nuclear run-on experiments have shown that the high level of expression of the mouse cytosolic glutathione peroxidase mRNA in erythroid cells is due to up-regulation of the gene at the transcriptional level. Studies of the chromatin structure around the cytosolic glutathione peroxidase gene have revealed a series of DNase I hypersensitive sites (DHSS) in the 3' flanking region of the gene in erythroid and other high-expression tissues that are lacking in low-expression cells, in addition to a DHSS over the promoter region in both high- and low-expression tissues. Functional transfection experiments have demonstrated that one of the 3' DHSS regions functions as an enhancer in erythroid cells but not in a low-expression epithelial cell line; and site-directed mutagenesis and footprinting experiments reveal that the activity of the erythroid cell-specific enhancer requires a cluster of binding sites for the CACC/GT box factors and the GATA and Ets families of transcription factors.

Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53.

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.