Abstract: PURPOSE: Clinical radiosensitivity varies considerably among patients, and radiation-induced side effects developing in normal tissue can be therapy limiting. Some single nucleotide polymorphisms (SNPs) have been shown to correlate with hypersensitivity to radiotherapy. We conducted a prospective study of 87 female patients with breast cancer who received radiotherapy after breast surgery. We evaluated the association between acute skin reaction following radiotherapy and 11 genetic polymorphisms in DNA repair genes: XRCC1 (Arg399Gln and Arg194Trp), XRCC3 (Thr241Met), XPD (Asp312Asn and Lys751Gln), MSH2 (gIVS12-6T>C), MLH1 (Ile219Val), MSH3 (Ala1045Thr), MGMT (Leu84Phe), and in damage-detoxification GSTM1 and GSTT1 genes (allele deletion). METHODS AND MATERIALS: Individual genetic polymorphisms were determined by polymerase chain reaction and single nucleotide primer extension for single nucleotide polymorphisms or by a multiplex polymerase chain reaction assay for deletion polymorphisms. The development of severe acute skin reaction (moist desquamation or interruption of radiotherapy due to toxicity) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for cumulative biologically effective radiation dose. RESULTS: Radiosensitivity developed in eight patients and was increased in carriers of variants XRCC3-241Met allele (hazard ratio [HR] unquantifiably high), MSH2 gIVS12-6nt-C allele (HR = 53.36; 95% confidence intervals [95% CI], 3.56-798.98), and MSH3-1045Ala allele (HR unquantifiably high). Carriers of XRCC1-Arg194Trp variant allele in combination with XRCC1-Arg399Gln wild-type allele had a significant risk of radiosensitivity (HR = 38.26; 95% CI, 1.19-1232.52). CONCLUSIONS: To our knowledge, this is the first report to find an association between MSH2 and MSH3 genetic variants and the development of radiosensitivity in breast cancer patients. Our findings suggest the hypothesis that mismatch repair mechanisms may be involved in cellular response to radiotherapy. Genetic polymorphisms may be promising candidates for predicting acute radiosensitivity, but further studies are necessary to confirm our findings.
Abstract: Recently, studies were conducted to evaluate the impact of T regulatory (T regs) cells in the pathophysiology of atopic dermatitis (AD). The aim of this study was to investigate whether natural T regs are present in AD skin lesions. We performed skin biopsies in 12 adult patients affected by moderate-to-severe AD and 4 healthy volunteers. The specimens were stained immunohistochemically with anti-human CD25 and forkhead/winged helix transcription factor (FoxP3). Double immunostaining for CD25 and FoxP3 was performed also. CD25+ cells strongly infiltrated the perivascular and papillar dermis of all lesional specimens, and FoxP3+ cells were distributed in the perivascular and interstitial AD dermis, and some cells also infiltrated the dermoepidermal junction and the basal and suprabasal epidermal layers. All healthy skin specimens showed weak CD25 and FoxP3 stainings. Double immunostaining showed that CD25+ FoxP3+ cells were distributed in the perivascular, interstitial, and periadnexal dermis, and healthy skin specimens featured few CD25+ FoxP3+ cells scattered throughout the dermis. The past and present data show that an impaired function of natural T regs may not play a primary role in the pathophysiology of AD lesions.
Abstract: BACKGROUND: Erythema multiforme (EM) and Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) are caused by a dysregulation of cellular immunity. OBJECTIVES: To evaluate further the potential role of certain cytokines and chemokine receptors in cutaneous lesions of patients affected by EM and SJS/TEN and to establish whether such diseases are polarized preferentially towards a T-helper (Th) 1 or Th2 pattern. METHODS: Biopsy specimens from eight patients with EM, six with SJS/TEN and three healthy controls were stained for immunohistochemical examination using the alkaline phosphatase-antialkaline phosphatase method. The monoclonal antibodies used included those to tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-2, IL-5, IL-13, receptor 3 for C-C chemokines (CCR3), receptor 3 for C-x-C chemokines (CXCR3) and CXCR4. RESULTS: The SJS/TEN specimens showed significantly higher expression of all the cytokines and chemokine receptors (except CXCR3) tested than the EM specimens. Both lesional series showed significantly higher expression of all the receptors tested than the healthy control specimens, with the sole exception of a lower expression of CCR3 in EM specimens. Comparison between molecules associated with a Th1 or Th2 response revealed a predominance of Th1 response in EM and no significant imbalance between Th1 and Th2 in SJS/TEN. CONCLUSIONS: We have provided further evidence that TNF-alpha is strongly expressed in SJS/TEN lesions and therefore it may be involved in the epidermal necrosis featured in such diseases. IFN-gamma may play an important role both in EM and SJS/TEN. IL-2, IL-5 and IL-13 may contribute to the cutaneous immunoinflammation in these diseases. Chemokine receptors may be involved strongly in the recruitment of inflammatory cells in lesional skin. In our cases we found a sharp polarization towards a Th1 pattern in EM, while the SJS/TEN lesions showed a mixed Th1/Th2 pattern.
Abstract: BACKGROUND: Erythema multiforme (EM) and Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) are determined by a dysregulation of cellular immunity. OBJECTIVES: To evaluate the effector role of cellular immunity and the involvement of the CD40/CD40 ligand (CD40L) system in the pathogenesis of EM and SJS/TEN. METHODS: Biopsy specimens from eight patients with EM and six with SJS/TEN were stained for immunohistochemical examination using the alkaline phosphatase/antialkaline phosphatase method. The monoclonal antibodies used included those to CD1a, CD4, CD8, CD40, CD40L, CD68, Fas, Fas ligand (FasL) and myeloperoxidase. RESULTS: The cellular infiltrate in both EM and SJS/TEN lesions was composed mainly of T lymphocytes and CD68+ macrophages. We also detected large amounts of neutrophils. Fas and FasL were very highly expressed in SJS and TEN, but weakly in EM. CD40 staining was strong in all tissue sections; there were numerous CD40L+ cells in SJS/TEN but much fewer in EM. CONCLUSIONS: Activated T lymphocytes and macrophages, but also neutrophils, are presumably the main triggers of mucocutaneous damage in the SJS/TEN disease spectrum. The Fas/FasL system is significantly expressed in SJS/TEN lesions, but not in EM, where this apoptotic pathway presumably does not play a pivotal role in the epidermal damage. We suggest that the CD40/CD40L system may represent an important pathway of induction of SJS/TEN lesions, while in EM it would contribute to the immunoinflammation only as a second-line mechanism.
Abstract: Apoptosis is a form of cell death that is claimed to be involved in a number of chronic inflammatory and malignant skin diseases. The aim of this study was to investigate whether apoptosis may contribute to the pathogenesis of epidermal changes in dermatitis herpetiformis (DH) and, in particular, whether certain apoptosis-related markers such as Bax, Bcl-2, Fas and Fas ligand (FasL) take part in this process. For the detection of apoptotic nuclei, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling technique (TUNEL) was employed on cryostat sections. Skin lesions from six and perilesional skin from four DH patients were stained with monoclonal antibodies to Bax, Bcl-2, Fas and FasL. The same evaluation was also performed on three patients affected by bullous pemphigoid (BP) and in two healthy donors. Using TUNEL technique, a remarkable increase in the apoptotic rate within the epidermal compartment was observed in DH and BP patients in comparison with normal controls. In our immunohistochemical analysis, Bax/Bcl-2 ratio was almost the same in the epidermis of perilesional/lesional DH, BP and healthy skin specimens. In DH and BP specimens both Bax and Bcl-2 proteins were increased in the dermal perivascular compartment. Fas showed a prevalently epidermal staining, both in DH and BP lesions, while FasL was distributed in perivascular and subjunctional dermis; some FasL+ cells infiltrated the DEJ and the basal layer of epidermis. This study allowed us to highlight conspicuous apoptotic phenomena in basal and suprabasal keratinocytes within lesional and perilesional skin of DH. We conclude that in DH, as well as in BP, apoptosis plays a role in the pathogenesis of cutaneous lesions in concert with other pathogenetic mechanisms.
Abstract: Mutations of the N- and K-ras genes occur in approximately 15-30% of acute myeloid leukaemia patients. The role of the oncogenic ras in leukaemogenesis remains unclear. Few studies have revealed that mutations in the ras oncogene family are more probably found in acute myeloid leukaemia patients with previous exposure to toxic agents. A case-case study was conducted in the areas of Florence and Turin, Italy, to investigate whether the presence of N- and K-ras mutations in acute myeloid leukaemia patients was related to a higher frequency of exposure to chemicals. During a 3-year period, 111 acute myeloid leukaemia patients were enrolled. All the patients were interviewed using a semi-structured questionnaire collecting data on residential history, occupation, personal habits and pathological history. The presence of N- and K-ras mutations was analysed by amplification and synthetic oligonucleotide probes and by the so-called polymerase chain reaction amplification for specific alleles technique. A total of 34 (30.6%) patients were found to harbour ras mutations in N-ras and/or K-ras. Fourteen patients (12.6%) had a single ras mutation and 20 patients (18%) had two ras mutations. A positive association between a priori at risk jobs and ras mutations was found, based on nine exposed cases; the odds ratio, adjusted by age, sex and previous X-ray and/or chemotherapy was 2.8 (95% confidence intervals: 0.9-9.0). When considering only subjects with two ras mutations the odds ratio was 4.8 (95% confidence intervals: 1.2-18.8). The odds ratio for a previous X-ray and/or chemotherapy was 16.2 (95% confidence intervals: 1.8-755.9); when only subjects with two ras mutations were considered, the odds ratio was 26.1 (95% confidence intervals: 2.5-1248.9). In conclusion, our data suggest that ras oncogene mutations might identify a group of leukaemia in people with previous X-ray/chemotherapy or with exposure to chemical agents in the work environment.
Abstract: Platelet Activating Factor (PAF), an inflammatory bioactive lipid, has been shown to be involved in the regulation of the activity of matrix metalloproteinases (MMPs). In view of the role played by MMPs in tumor cell invasiveness, we investigated whether PAF influences MMP activity in a system of neuroblastoma clones, the AA5 and AE12 cells, isolated from the human LaN1 neuroblastoma cell line. These clones were characterized by an inverse relationship between invasiveness and differentiative capacity and by the expression of specific cell surface PAF receptors. We found that the levels of mRNAs specific for MMP-2 and for MT1-MMP, the MMP-2 activator, were reduced in both clones treated with 300 nM PAF. These changes are consistent with the reduced secretion and activation of MMP-2 found in the neuroblastoma clones exposed to PAF. These effects were accompanied by an inhibition of invasiveness through Matrigel and by a promotion of differentiation, as revealed by an increased percentage of cells with neurites. The finding that both neuroblastoma clones exposed to the metalloproteinase inhibitors, BB3103 and 1,10-phenanthroline, increased their differentiative capacity and reduced their invasiveness through Matrigel, represents a further indication that PAF modulates differentiation and invasiveness by affecting the activity of MMPs.
Abstract: Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14C-acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography-mass spectrometry with a negative ion chemical ionization. In the presence of the Ca2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFalpha and IFNgamma might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca2+ ionophore. Synthesis of PAF was not inducible by TNFalpha in murine F10-M3 melanoma cells. IFNgamma also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.
Abstract: The effect of CNTF and BDNF on a proteolytic complement instrumental to invasion and on differentiation was studied in two murine neuroblastoma clones, N1 and N7. At the membrane level, gelatinase MMP-2--mainly the activated form--was restrained by CNTF and BDNF to a residual 34% with both factors; membrane-type 1 MMP was down-regulated to 50% (10 h) and 34% (24 h) with both factors; and urokinase-type plasminogen activator was restrained mainly by BDNF to 70%. In the medium, the two gelatinases MMP-2 and MMP-9 were mainly in zymogen form: only MMP-2 was restrained in N1 cells, while only MMP-9 was restrained in N7 cells by both factors, single or in combination. These effects were paralleled by the induction of neurite outgrowth, which was more stimulated in the less differentiated clone. These dose-dependent and transient effects make CNTF and BDNF ideal candidates for constraining the potentially invasive behavior of nervous system tumors.
Abstract: The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti-u-PA and anti-u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA-induced invasion, as well as in u-PA-induced proliferation.
Abstract: We investigated whether tumor cell/endothelia interaction can be influenced by platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a lipid mediator that promotes adhesiveness and extravasation of leukocytes in the inflammatory reaction. We found that the PAF receptor antagonist WEB 2086 prevents adhesion of melanoma Hs294T and colon carcinoma LS180 lines to IL-1-stimulated endothelial cells. Moreover, PAF stimulated the adhesiveness of Hs294T and LS180 cells to VCAM-1 and E- selectin, respectively, in an artificial model consisting of recombinant adhesive proteins bound to protein A-coated substrata. Thus, tumoral and not endothelial cell surface seems to be involved in the PAF-mediated enhancement of tumor cell adhesiveness to IL-1-activated endothelia. This observation is supported by the finding that Hs294T and LS180 cells express high affinity and functionally active receptors for PAF. By using specific inhibitors, we found that PAF-induced enhancement of cell adhesiveness was mediated by G-protein activation and protein tyrosine phosphorylation. In addition, protein tyrosine phosphorylation was observed in Hs294T and LS180 cells stimulated by PAF. In conclusion, we demonstrated that PAF-mediated activation of tumor cells enhances their adhesiveness to IL-1-stimulated vascular endothelia.
Abstract: Interest in lipid characteristics of metastatic cells was aroused by the consideration that the various lipid components of cell membranes influence a broad spectrum of cell surface biological functions which are involved in different steps of the metastatic cascade. Correlation between invasive properties and characteristics of cell surface components has been appropriately studied in a limited number of metastatic cell systems isolated by in vivo and in vitro procedures. The major findings of this study have been reported in this review. Among membrane lipid components, glycolipids and phospholipids appeared particularly affected in tumor cells which acquired a metastatic phenotype. In fact, the reduction of complex gangliosides typical of transformed cell lines was even more evident in a highly metastatic variant selected from RSV-transformed murine fibroblasts. The reduction of complex gangliosides, mainly GD1a, particularly affected the adhesion sites of this variant. In a fibrosarcoma line, T3 cells, the metastatic properties appeared to be correlated with the content and cell surface expression of Gb3ose, a glycolipid characteristic of this line. Moreover, a particularly high level of ether-linked lipids was found in high metastatic variants isolated from murine melanoma and fibrosarcoma lines, as well as in human mammary carcinomas. The findings considered in this review are discussed for their possible relevance to the invasive properties of metastatic cells.
Abstract: In order to clarify the relationship between invasiveness and loss of cellular differentiation in tumor cells, we studied the invasive properties on Matrigel of (a) a series of clones we isolated from human neuroblastoma LaN1 and Platt cell lines inducible to differentiation by adhesion on fibronectin, and (b) SY5Y human neuroblastoma cells inducible to differentiation by retinoic acid. We found that, regardless of the parental line, the more differentiated clones were scarcely invasive, while the less differentiated clones showed a higher degree of invasiveness. Differences in invasiveness between differentiated and non-differentiated neuroblastoma clones did not reflect differences in adhesiveness to laminin, the major component of Matrigel. The retinoic acid-sensitive SY5Y human neuroblastoma cells also reduced their invasiveness on Matrigel after differentiation induced by growth in media supplemented with retinoic acid. These results point to an inverse relationship between differentiative properties and invasiveness in human neuroblastoma cell lines.
Abstract: In the past few years levels of exposure to environmental agents have lowered and people are often exposed to many different chemicals and mixtures. Therefore, it has become much more difficult to establish relationships between exposure and disease by traditional methods of epidemiology. Molecular epidemiology was developed to overcome the problem, and biochemical or biological markers were used to increase the power of environmental epidemiology. This paper summarizes the most important literature about applications of biological markers in epidemiology.
Abstract: In this study, we investigated whether complex gangliosides influence cell adhesion by modulating the activity of integrin receptors. Our experimental model was represented by CMH5123 cells, a line of neoplastic hepatocytes derived from the minimal deviation Morris hepatoma 5123c of the rat, which adhered to substrata coated with fetal calf serum (FCS) by an integrin-mediated mechanism, being vitronectin the specific serum protein which sustained cell adhesion. We found that ganglioside depletion, obtained by inhibiting complex ganglioside biosynthesis, was accompanied by a reduction of cell adhesiveness to FCS-coated substrata. Integrins appeared to mediate the effect of ganglioside depletion on cell adhesiveness. In fact, sensitivity to the integrin inhibitor GRGDSPC peptide was ten times higher in ganglioside-depleted cells compared to control cells. Moreover, growth of ganglioside-depleted CMH5123 cells in media supplemented with complex gangliosides restored the cell sensitivity to the integrin inhibitor to the same level as that found in control cells. Furthermore, ganglioside depletion of CMH5123 cells decreased the affinity of vitronectin receptors for vitronectin without modifying their number, affinity of vitronectin receptors was re-established in ganglioside-depleted cells by supplementing their growth media with complex gangliosides. In conclusion, these results support the participation of gangliosides to cell adhesion as modulators of integrin receptors.
Abstract: Electrical signals elicited by integrin interaction with ECM components and their role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN), this adhesion induced neurite outgrowth in N1 but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), N1 cells undergo a marked (approximately equal to 20 mV) hyperpolarization of the resting potential (Vrest) that occurred within the first 20 min after cell contact with ECM, and persisted for approximately 1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptor (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on Vrest of N7 cells, abolished in N1 cells the FN- or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the alpha V containing complexes. Both clones possessed Ca2+ and K+ delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K+ (KIR) channels. The latter governed the Vrest, and, unlike KDR channels, were blocked by Ba2+ and Cs+. By moving patched cells in contact with FN-coated beads, it was shown that KIR channel activation was responsible for the FN-mediated hyperpolarization of Vrest. Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G protein is interposed between integrins and KIR channels and that the activation of these channels is required for neuritogenesis. In fact, the block of KIR channels by Cs+ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the first two hours after N1 cell contact with FN.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: Subclones of F11 neuronal hybrid cells (neuroblastoma x dorsal root ganglion neurons) have segregated differing and/or overlapping neuritogenic mechanisms on three substrata--plasma fibronectin (pFN) with its multiple receptor activities, cholera toxin B subunit (CTB) for binding to ganglioside GM1, and platelet factor-4 (PF4) for binding to heparan sulfate proteoglycans. In this study, specific cell surface receptor activities for the three substrata were tested for their modulation during neuritogenesis by several experimental paradigms, using F11 subclones representative of three differentiation classes (neuritogenic on pFN only, on CTB only, or on all three substrata). When cycloheximide was included in the medium to inhibit protein synthesis during the active period, neurite formation increased significantly for all subclones on all three substrata, virtually eliminating substratum selectivity for differentiation mediated by cell surface integrin, ganglioside GM1, or heparan sulfate proteoglycans. Therefore, one or more labile proteins (referred to as disintegrins) must modulate functions of matrix receptors (e.g., integrins) mediating neurite formation. To verify whether cycloheximide-induced neuritogenesis was also regulated by integrin interaction with cell surface GM1, two approaches were used. When (Arg-Gly-Asp-Ser)-containing peptide A was added to the medium, it completely inhibited cycloheximide-induced neuritogenesis on all three substrata of all subclones, indicating stringent requirement for cell surface integrin function in these mechanisms. In contrast, when CTB or a monoclonal anti-GM1 antibody was also added to the medium, cycloheximide-induced neuritogenesis was amplified further on pFN and sensitivity to peptide A inhibition was abolished. Therefore, in some contexts ganglioside GM1 must complex with integrin receptors at the cell surface to modulate their function. These results also indicate that (a) cycloheximide treatment leads to loss of substratum selectivity in neuritogenesis, (b) this negative regulation of neurite outgrowth is affected by integrin receptor association with labile regulatory proteins (disintegrins) as well as with GM1, and (c) complexing of GM1 by multivalent GM1-binding proteins shifts neuritogenesis from an RGDS-dependent integrin mechanism to an RGDS-independent receptor mechanism.
Abstract: Clones of F11 hybrid (neuroblastoma X dorsal root neuron) cells have been tested for adherence and neurite outgrowth on three different substrata on which the parental cells display some competence--plasma fibronectin (pFN) with its multiple receptors, cholera toxin subunit B(CTB) as a model ganglioside GM1-binding substratum, and platelet factor-4 (PF4) as a model proteoglycan-binding substratum. This paradigm tests for independently segregating and overlapping mechanisms of neuritogenesis via transmembrane processes in pluripotent hybrid cells based on random loss of chromosomes contributed by the parent neural cells. For the nine clones tested, attachment was significantly lower on CTB but much higher on PF4 for all clones when compared to their attachment on pFN. Supplementation of cells with GM1 stimulated attachment of only two clones (on all three substrata). Neurite outgrowth was observed in a substratum-specific pattern and varied from 0 to greater than 60% on pFN; on CTB and PF4 substrata, the patterns were similar to each other but differed markedly from the pattern on pFN. In contrast, PF4- and CTB-directed neurites differed morphologically from each other while sharing some characteristics with neurites on pFN. Supplementation with GM1 or GT1b, but not GD1a, was inhibitory for neurite outgrowth in certain clones. Cycloheximide pretreatment distinguished several classes of clones based on inhibition of neuritogenesis. While most clones on pFN were unaffected, all clones on CTB and PF4 displayed significant and comparable degrees of inhibition, suggesting the sharing of some protein intermediate(s) on these substrata. Exposure to cycloheximide only during the active period of neuritogenesis generated higher percentages and longer neurites for all clones, indicating a widely-used negative regulation mechanism. Based on substratum type and cycloheximide protocols, these data have resolved six or more different transmembrane signalling processes for generating different classes of neurites. Some mechanisms have been segregated into individual clones while others overlap in other clones, providing cell systems for biochemical and molecular biological dissection of these processes.
Abstract: This study compares the ganglioside composition of tissue culture substrate-attached material (SAM) with that of cell bodies in a line of transformed hepatocytes derived from the minimal deviation Morris hepatoma 5123 c (CMH5123 cells). We examined both confluent cultures (late-phase cultures) and cells which were allowed to attach for only 3 h (early-phase cultures). We also determined to what extent ganglioside compositions of SAM and cell bodies from early- and late-phase cultures of CMH5123 cells are affected by the block of complex ganglioside biosynthesis induced by treatment with chelating agents (EGTA + EDTA). The morphological characteristics of SAM were monitored by scanning electron microscopy during the different steps of this study. In early-phase cultures, SAM was composed of fragments of filopodia and small vesicles probably representing newly formed substratum adhesion sites. In contrast, SAM of late-phase cultures was made up of large pools of membranous material resulting from the breakage of thick retraction fibers connecting the cell body with broad, mature adhesion sites. SAM of early-phase cultures yielded ganglioside profiles with a higher content of GM1 and GD1 a than those of cell bodies, while in late-phase cultures there was no difference between SAM and cell body gangliosides. When cells were grown in the presence of chelating agents, SAM of early-phase cultures was composed of vesicles and filopodial fragments similar to those found in early-phase cultures grown in regular media; these morphological features also appeared in SAM of confluent cultures (in contrast to the membranous material characteristic of late-phase cultures grown in regular media). In early-phase cultures grown in the presence of chelating agents, gangliosides of SAM were enriched in complex homologs relative to their content in cell bodies. These ganglioside characteristics were also found in SAM of confluent cultures grown in the presence of chelating agents, reflecting the presence of newly formed adhesion sites. On the basis of these results, we may conclude that the molecular assembly of newly formed adhesion sites implies the preferential distribution of several surface components involved in cell adhesion, including complex gangliosides.
Abstract: To determine whether a metastatic phenotype may be correlated with a characteristic lipid pattern, we compared the lipid composition of low metastasizing Balb/c 3T3 cells transformed by the B77 strain of Rous sarcoma virus (B77-3T3 cells) with that of a subclone isolated by growth in 0.6% agar, the B77-AA6 cells, which exhibit a high capacity for spontaneous metastasis. B77-3T3 cells revealed characteristics in their lipid composition common to other systems of transformed cells, i.e., an accumulation of ether-linked lipids, a reduction of the more complex gangliosides, an increase of oleic acid (18:1) and a decrease of arachidonic (20:4) and C22 polyunsaturated fatty acids in phospholipids. High metastatic B77-AA6 cells showed: a) an even more marked decrease of complex gangliosides; b) a more pronounced increase of 18:1 and decrease of 20:4 and 22 polyunsaturated fatty acids in certain phospholipid classes; and c) a higher percentage of alkyl-acyl subfractions in both phosphatidylcholine and phosphatidylethanolamine than B77-3T3 cells. Comparing the data for other systems of metastatic cells with those of lipid studies of spontaneously metastasizing B77-AA6 cell system leads us to conclude that the metastatic phenotype is characterized by a change in ether-linked lipids, rather than in fatty acids.
