Detection of putative stem cell markers, CD44/CD133, in primary and lymph node metastases in head and neck squamous cell carcinomas. A preliminary immunohistochemical and in vitro study.

OBJECTIVES: Investigators hypothesized that cancer stem cells (CSCs) could play a role in determining cancer progression by metastasizing to cervical lymph node (N+) and then influencing prognosis of head and neck squamous cell carcinomas (HNSCCs) patients. DESIGN: To identify CSCs in HNSCCs and their clonogenic capacity. SETTING: In vitro study. PARTICIPANTS: Putative CSCs from 29 primary HNSCCs and 19 corresponding node metastases were analyzed. MAIN OUTCOME MEASURES: Immunohistochemical (IHC) was performed, and CSCs' clonogenic in vitro capacity was tested; ones epithelial nature of cancer cells forming colonies was confirmed by a second IHC, fluorescence-activated cell sorting (FACS) analysis helped in counting CD44/CD133-CSCs markers percentage expression in HNSCC tumour-derived cultures. RESULTS: Immunohistochemical showed CD44 (93.1%) and CD133 (10.34%) expression; FACS-analysis showed the enrichment of CD44/CD133 cancer cells, with the highest clonogenic capacity of CD44+-subpopulation; a higher CD44 rates were documented from N+ subcultures than from original tumours (P < 0.05). CONCLUSIONS: A putative cancer stem-like cell population is detectable in HNSCCs, and our findings show their in vitro clonogenic capacity by demonstrating that CD44+-cultured cells are the main population proliferating obtained by N+ HNSCC metastases, emphasizing their possible role in tumour progression.

Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic death of bystander cells.

An emerging strategy for cancer gene therapy involves the transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene into tumor cells, rendering them susceptible to the cytotoxic effects of ganciclovir. The observation that HSV-tk-expressing cells can also induce cell death in neighboring cells, which do not express HSV-tk, has been called the bystander effect. Gap junction-mediated transfer of cytotoxic molecules to bystander cells may be an important mechanism of bystander cell death, although others have suggested a role for phagocytosis. In this study, we evaluated the mode of cell death in bystander cells. We detected apoptosis in bystander cells and found that bystander cell death could be inhibited by BCL2 expression. We determined that ganciclovir incubations for 10 h were sufficient to induce cell death in most bystander cells cocultured with HSV-tk-expressing cells. During this period, no phagocytosis was detected, although it was obvious at later stages.

Polyphosphoinositide metabolism is rapidly stimulated by activation of a temperature-sensitive mutant of Rous sarcoma virus in rat fibroblasts.

We have examined polyphosphoinositide turnover in a Rat-1 fibroblast line infected with a temperature-sensitive mutant (ts LA24) of the Rous sarcoma virus (RSV). When ts LA24-infected cells are shifted from the non-permissive to the permissive temperature, a rapid and sustained activation of phospholipase C (PLC) is observed. Normal and wild-type RSV-infected Rat-1 cells do not show any PLC activation upon temperature shiftdown. Pre-treatment of ts LA24-infected fibroblasts with tetrodotoxin (a Na+ channel inhibitor) or incubation in Na+-free medium significantly prevent temperature shiftdown-induced PLC activation. Therefore, we conclude that PLC activation occurs concomitantly with pp60v-src expression, and hypothesize that pp60v-src-related membrane depolarization is the causal link between pp60v-src tyrosine kinase activity and stimulation of polyphosphoinositide metabolism. Finally, we discuss the relationship between the phenomena we have observed and the mechanism of action of the ras oncogene.

Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer. Implications for tumor angiogenesis and metastasis.

We evaluated the role of COX-2 pathway in 35 head and neck cancers (HNCs) by analyzing COX-2 expression and prostaglandin E2 (PGE2) production in relation to tumor angiogenesis and lymph node metastasis. COX-2 activity was also correlated to vascular endothelial growth factor (VEGF) mRNA and protein expression. COX-2 mRNA and protein expression was higher in tumor samples than in normal mucosa. PGE2 levels were higher in the tumor front zone in comparison with tumor core and normal mucosa (P<.0001). Specimens from patients with lymph node metastasis exhibited higher COX-2 protein expression (P=.0074), PGE2 levels (P=.0011) and microvessel density (P<.0001) than specimens from patients without metastasis. A significant correlation between COX-2 and tumor vascularization (r(s)=0.450, P=.007) as well as between COX-2 and microvessel density with VEGF expression in tumor tissues was found (r(s)=0.450, P=.007; r(s)=0.620, P=.0001, respectively). The induction of COX-2 mRNA and PGE2 synthesis by EGF and Escherichia coli lipopolysaccharide (LPS) in A-431 and SCC-9 cell lines, resulted in an increase in VEGF mRNA and protein production. Indomethacin and celecoxib reversed the EGF- and LPS-dependent COX-2, VEGF, and PGE2 increases. This study suggests a central role of COX-2 pathway in HNC angiogenesis by modulating VEGF production and indicates that COX-2 inhibitors may be useful in HNC treatment.

Role of specific membrane receptors in urokinase-dependent migration of human keratinocytes.

On the basis of both 125I-labeled plasminogen activator binding analysis and transmission electron microscopy studies of the interaction of a plasminogen activator/gold complex with cell membranes, we have found that human keratinocytes have specific receptors for human urokinase-type plasminogen activator distributed on the cell surface as singlets, or as small or large clusters. The use in binding experiments of the purified A chain of urokinase-plasminogen activator and of anti-A chain monoclonal antibodies has indicated that cell receptors are specific for a sequence present on the A chain, as previously reported for other cells. The interaction of both the native molecule and the purified A chain with such receptors stimulates mobilization of keratinocytes in an in vitro cell model system (Boyden chamber), when present in the lower compartment of the migration apparatus in nanomolar concentrations. Preincubation of chemoattractants with a monoclonal antibody which prevents receptor/ligand interaction also prevents plasminogen activator-induced cell migration. These data suggest that, under the conditions used in this in vitro model system, the plasminogen activator-dependent mobilization of keratinocytes depends on the interaction of the ligand with free receptors on the cell surface, and is independent of plasmin generation.

Signal transduction in EJ-H-ras-transformed cells: de novo synthesis of diacylglycerol and subversion of agonist-stimulated inositol lipid metabolism.

We examined the level of 1,2-diacylglycerol and inositol phosphates in normal and EJ-H-ras-transformed BALB/3T3 fibroblasts by prelabelling the cells with [3H]glycerol, [3H]inositol, [14C]glucose, [14C]arachidonic acid, and [14C]palmitic acid. Steady-state level of inositol phosphates, however, was the same in control and transformed cells. Diacyglycerol labelling by [14C]arachidonic acid was the same in control and transformed cells. Insulin dramatically increased diacylglycerol labeling by [14C]glucose in normal cells, whereas it did not affect ras-transformed fibroblasts. Neurotransmitter-induced inositol lipid turnover was greatly enhanced in ras-transformed cells; conversely, platelet-derived growth factor and thrombin-stimulated normal cells to a greater extent than transformed fibroblasts. Taken together these results suggest that ras transformation may induce multifarious effects on signal transduction: it may cause de novo synthesis of diacylglycerol and subversion of neurotransmitter and growth factor receptor coupling to inositol lipid metabolism.

Negative growth control by a novel low M(r) phosphotyrosine protein phosphatase in normal and transformed cells.

Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.

Plasminogen activators and tiaprofenic acid in inflammation. A preliminary study.

Treatment with tiaprofenic acid appreciably reduced the level of plasminogen activators in the medium of 3T3-Balb mouse fibroblasts, as revealed by both a fibrin plate assay and amidolytic determination with chromogenic substrates. At the same time, tiaprofenic acid was able to inhibit the production of plasminogen activators induced by phorbol myristate acetate, a powerful inflammation and tumour promoter, added to the cell monolayers. By isolating the inhibitors of plasminogen activators it was possible to show that the decrease of fibrinolytic activity produced by tiaprofenic acid is not related to an increase of inhibitors. Rather, a decrease of activators seems to take place. Synovial fluid samples from 4 patients before and after treatment with tiaprofenic acid were also assayed for plasminogen activator activity by the fibrin lysis method. In 3 of the 4 cases a marked decrease after treatment was evident. The one unresponsive patient suffered from a para-neoplastic arthritis.

In vitro modulation of MCF-7 breast cancer cell growth by myoepithelial cells.

Growth and differentiation of MCF-7 breast adenocarcinoma cells were studied in mixed cultures of MCF-7 cells and PA 16/23 myoepithelial cells and in isolated cultures of MCF-7 cells grown in the absence and presence of conditioned medium of PA 16/23 cells. In the cocultures, the MCF-7 cells grew in smaller aggregates and showed a more differentiated phenotype than in the isolated cultures. The conditioned medium of PA 16/23 cells enhanced growth and reduced differentiation of the MCF-7 cells. Hence, a direct relationship between MCF-7 cells and PA 16/23 cells seems to play a leading role in influencing the behaviour of the former cells, thus overriding the opposite effects of soluble factors secreted by the PA 16/23 cells.

Binding, internalization and degradation of heparin and heparin fragments by cultured endothelial cells.

We have studied the binding to bovine adrenal capillary endothelial cells cultured in vitro of heparin from different sources (porcine heparin Ep. 152 P, Av.M.W. 15.9 Kd and bovine heparin Ep. 756 P, Av.M.W. 12.9 Kd) and heparin fractions of various molecular weights (low molecular weight heparin, LMW 2123 OP, Av.M.W. 4.5 Kd and very low molecular weight heparin, VLMW 1027/45 OP Av.M.W. 2.1 Kd). The binding was specific for heparin; heparan sulphate showed some competition whereas dextran sulphate and glycosaminoglycans did not. We determined the affinity of heparin and heparin fragments for endothelial cells by means of displacement of bound 3H-labeled heparin in response to increasing concentration of unlabeled compounds. The binding of the different heparin fractions depends on their molecular weights. VLMW 1027/45 OP was unable to bind to the cells, whereas LMW 2123 OP showed an affinity 10 times lower then porcine heparin. Bovine adrenal capillary endothelial cells incubated with unfractionated 3H-labeled heparin selectively bound internalized and degraded high molecular weight heparin fractions, as shown by gel filtration of the 3H-labeled heparin both after binding to the cells and after internalization.

Interaction between endogenous circulating sulfated-glycosaminoglycans and plasma proteins.

Interaction between endogenous 35S-labelled plasma glycosaminoglycans and proteins in murine plasma was demonstrated by coelution from gel chromatography of circulating 35S-labelled glycosaminoglycans with a wide range of plasma proteins. Autoradiography of electrophoretic tracing of proteins from 35S-sulfate labelled plasma showed that labelled glycosaminoglycans were associated with alpha 1, alpha 2, beta globulins and albumin, but not with gamma globulins. Analysis by gel chromatography on Sepharose CL-6B of delipidated 35S-labelled plasma after either proteolysis or beta-elimination, suggested that 35S-labelled glycosaminoglycan chains were covalently bound to proteins. Lipids were probably involved in the supramolecular assembly of GAGs with plasma proteins, as shown by hydrophobic interaction chromatography. In addition, strong, non-covalent interaction between glycosaminoglycan chains and proteins was responsible for the difficulty in extracting 'free' glycosaminoglycans from plasma. Consistently, ion-exchange chromatography of 35S-sulfate labelled delipidated plasma after alkali treatment, revealed that the anionic properties of glycosaminoglycans were hampered when plasma proteins were present.

Molecular polarity in endothelial cells and tumor-induced angiogenesis.

Endothelial cells expose receptors for vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) at the abluminal, basal surface that work as basic regulators of tumor-induced angiogenesis. Their specific localization makes them susceptible to the activity of tumor-released stimulatory factors, like VEGF/VPF, which induce proliferation of the endothelial cell toward the extracellular matrix. At the same time, VEGF/VPF stimulates endothelial cells to expose tissue factor (TF), the high-affinity transmembrane receptor and cofactor for cellular initiation of the plasma coagulation protease cascades through the extrinsic pathway, so generating thrombin. Thrombin exerts a number of activities: it forms an extracellular fibrin barrier from the VEGF/VPF-dependent fibrinogen extravasation; it activates progelatinase-A (pro-MMP-2), which destroys the basal membrane, allowing proliferation of endothelial cells (ECs) in the novel tumoral fibrin matrix; finally, it induces EC proliferation, potentiating the VEGF effect. Another important factor exposed at the abluminal endothelial cell surface is membrane type 1 matrix metalloproteinase (MT1-MMP), a membrane-bound metalloproteinase, which also activates progelatinase-A, allowing an alternative pathway to that of thrombin to destroy the basal membrane. In addition, we will see that MT1-MMP is also engaged in a direct, cell-associated fibrinolytic activity, essential for tubulogenesis of the novel outsprouting capillary.

Acetylation and phosphorylation of the carboxy-terminal domain of p53: regulative significance.

Three fundamental domains are conventionally distinguished on the p53 molecule: an NH2 domain involved in transcription, a central core domain involved in specific DNA binding to the consensus sequences, and a carboxy-terminal domain of about 30 amino acids working as a basic regulatory domain, exhibiting aspecific DNA binding, tetramerization, and nuclearization. The presence of an unmodified carboxy-terminus does not allow the specific transactivation transcriptional function of the p53 protein. Therefore, for the activation of the protein function the carboxy-terminus must be modified. In the present commentary we discuss the role of two covalent modification events occurring at the carboxy-terminus, namely phosphorylation and acetylation, as well as the specific role of these events in the functional regulation of p53 molecule.

Cox-2, iNOS and p53 as play-makers of tumor angiogenesis (review).

Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid to prostaglandins (PGs) and other eicosanoids. Nitric oxide synthase (NOS) is the enzyme that catalyzes the formation of nitric oxide (NO), a regulator of vascular permeability, from the guanidino nitrogen atom of L-arginine. Two isoforms of both enzymes occur: a constitutive one, Cox-1 and the inducible counterpart Cox-2; also NOS has a constitutive counterparts (cNOS) and an inducible form, called iNOS. The inducible isoforms of both enzymes are of maximum interest. It has been recently shown that cyclooxygenase-2 (Cox-2) is inducible by a variety of stimuli and that eicosanoids, mainly of the PGE2 species, are inducers of basic regulator of angiogenesis, including VEGF/VPF, bFGF, TGF-beta, PDGF, and endothelin-1. In addition, iNOS is inducible by Cox-2. p53 down-regulates the angiogenic process at various levels: it induces thrombospondin-1, a powerful antiangiogenic factor, down-regulates VEGF and NOS and, in addition, down-regulates hypoxia-induced angiogenesis, either inducing apoptosis or enhancing antiangiogenetic factors. It is noteworthy how important the p53 oncosuppressor is in the angiogenesis of solid tumor growth. Cox-2, iNOS and p53 are thus fundamental play-makers of the angiogenic process: they are discussed in detail and a tentative hierarchical cascade is proposed.

Apoptosis : molecular regulation of cell death and hematologic malignancies.

Apoptosis, or programmed cell death, represents in cell biology a functional program as important as cell growth or differentiation. Programmed cell death is of basic importance for the development of multicellular organisms and its basic mechanisms are conserved during the evolution of metazoa. Mammalian cells exhibit several different apoptotic pathways that converge to a common endpoint. Each pathway is triggered by a different stimulus: growth factor default, irradiation, induction of the p53 oncosuppressor protein, glucocorticoid hormones (in lymphocytes), ligand binding to Fas/APO (CD95), or tumor necrosis factor receptor (TNF-R), perforin secreted by cytotoxic T cells (reviewed by Hale et al. [1]). As opposed to necrosis, apoptosis is a "clean" process: as the cell shrinks, the cell membrane turns into the "apoptotic shell," the nucleus is condensed and reduced in volume, and eventually the cell disappears from the tissue, due to phagocytosis by neighboring cells or professional phagocytes, such as macrophages.

Inhibition of spontaneous growth and induced differentiation of murine erythroleukaemia cells by paraquat and atrazine.

The effect of the herbicides paraquat and atrazine on erythroid differentiation has been studied in mouse erythroleukaemic cells. The addition of atrazine or paraquat was shown to inhibit both spontaneous growth and hexamethylene-bis-acetamide (HMBA)-induced differentiation of undifferentiated erythroleukaemic cells. This effects was dose-dependent and occurred at concentrations of less than 10 ppm for both herbicides. Growth inhibition with atrazine (40-45%) was less pronounced than with paraquat (85-90%). Inhibition of differentiation paralleled growth inhibition. A synergistic effect was observed with HMBA, which per se reduced the growth rate of mouse erythroleukaemic cells, and either herbicide. Evaluation of cell viability under all the experimental conditions using either a trypan blue dye exclusion test or labelled chromium indicated that the effects observed were not related to a cytocidal action of atrazine or paraquat.

Craniofacial development in rats with early resection of the zygomatic arch.

Eighty-two-day-old male Wistar rats were selected to study the pattern of craniofacial growth following resection of the zygomatic arches. Rats were divided into three groups: group I (n = 14), the control group; group II (n = 15), with unilateral resection of the zygomatic arch; and group III (n = 8), with bilateral resection. Direct dry skull and cephalometric measurements show increased facial projection and decreased transverse facial width on the side of the resected arch. If the results are extrapolated to the growth pattern of patients with the Treacher Collins syndrome, we can conclude that the zygomatic arch acts as a "moderator" in the morphologic development of the face.

Role of p53 mutations in the radiosensitivity status of tumor cells.

Wild-type p53 is involved in cellular response to DNA damage including cell cycle control, DNA repair and activation of apoptosis. Accumulation of p53 protein following DNA damage may initiate the apoptotic process, resulting in cell death. DNA damage induced by radiation is an example of apoptotic stimulus involving p53. Regulation of apoptosis by p53 can occur through transcriptional regulation of pro-apoptotic (e.g. bax) and anti-apoptotic (e.g. bel-2) factors. Although wild-type p53 usually sensitizes cells to radiation therapy, p53 mutations have a variable effect on radiation response. For example p53 mutations in bone or breast tumors have been found to be associated with resistance to chemotherapeutic drugs or ionizing radiation. Mutated p53 has has been reported to increase sensitivity to radiation and drugs in colorectal and bladder tumors. The present brief commentary tries to find an explanation at molecular level of these conflicting results.

Cytokine receptor signal transduction mechanisms in immuno-hematopoietic cells.

Novel aspects of cytokine receptor signal transduction are discussed and cytokine receptors classified based on ligand-dependent signalling. An introductory section presents an overview of the role of cytokines in hematopoiesis. A brief explanation of basic concepts, such as redundancy, pleiotropy, synergism, etc., important for the understanding of cell response to cytokines, is also included. Three of five classes of receptors show the involvement of tyrosine kinase activity as the key step in signal transduction. The importance of tyrosine phosphorylation in cellular response to cytokines is pointed out.