Programmed cell death and mammary neoplasia.
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Animals↗
Biomedical subjects
R Strange
Publications and source records attributed to R Strange.
Glutathione S-transferase and cytochrome-P-450 polymorphism as risk factors for squamous cell carcinoma of the larynx.
PURPOSE: While cigarette smoking and alcohol consumption are recognized covariates for laryngeal carcinoma, the role of genetic factors in determining individual susceptibility is unknown. We describe the influence of polymorphism in carcinogen-metabolizing enzymes on susceptibility to squamous cell carcinoma of the larynx. MATERIAL AND METHODS: We investigated 269 patients with T1-T4 laryngeal carcinoma and 216 controls. Enzyme genotypes at the glutathione-S-transferase GSTM1 (A, B, A/B, null), GSTM3 (A, B), GSTT1 (null and expressors), and cytochrome P-450, CYP2D6 (intron 3/exon 4 boundary mutation and exon 5 deletion), CYP1A1 (3'-mutation and exon 7 mutation), and CYP2E1 (mutation at the 5' flanking region) loci were determined using polymerase chain reaction and restriction-based approaches. RESULTS: While the frequencies of the heterozygote GSTM1 A/B and homocygote GSTM3 B/ B were statistically significantly lower in cases than controls, the frequency of the GSTT1 null genotype was higher in the cases than controls. Genotype frequencies of 123 patients suffering squamous cell carcinomas located at different sites within the upper aerodigestive tract showed no differences between cases and controls. CONCLUSIONS: The data provide evidence that susceptibility to laryngeal carcinoma, but not pharyngeal carcinoma, is mediated by allelism at a number of loci encoding enzymes involved in the detoxification of electrophils derived from environmental pollution including cigarette smoke.
Carcinoma, Squamous Cell↗
Polymorphism in detoxifying enzymes and susceptibility to skin cancer.
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Antioxidants↗
Use of the yeast two-hybrid system for identifying the cascade of protein interactions resulting in apoptotic cell death.
Use of the yeast two-hybrid system allows rapid identification of interacting protein or proteins for a specific target protein. The technique is readily applied and allows immediate isolation of a cDNA encoding the interacting protein. One consideration might be to outline criteria for continued study of the interactors once they are identified. Our criterion for further study of an interactor is its presence in the mammary gland at a developmental time when the target protein is also present. Further characterization of interactors may involve immunoprecipitation, enzyme assays, or other techniques applicable to the specific protein.
Animals↗
Programmed cell death during mammary gland involution.
Understanding the cascade of gene expression and subsequent protein interactions that result both in the death of secretory mammary epithelium and the remodeling and renewal of the mammary gland for another cycle of lactation poses significant challenges (see Chapters 7 and 8, this volume). The complexity of mammary gland involution warrants caution in sorting through the various potential regulators and executors of apoptotic cell death in the mammary gland. As demonstrated by the number of remodeling enzymes expressed during involution, the relationship between mammary epithelium and its related mesenchyme is important for maintenance of differentiated function (Barcellos-Hoff et al., 1989; Streuli et al., 1991). Components of the extracellular matrix may play the role of survival factors, or may provide a source of factors, as a reserve of matrix-bound growth factors, necessary for survival of the secretory epithelium. Perturbation of this interaction alters mammary-specific differentiation gene expression, for example, production of milk proteins (Parry et al., 1987; Strange et al., 1991; Talhouk et al., 1992). Thus, alteration of the interaction between epithelium and its associated mesenchyme, which is an integral part of mammary involution, may also play a role in epithelial cell death. However, the epithelial-mesenchymal interactions that are the determining features in either mediating or modulating this cell death are just beginning to be defined. Stimuli that alter differentiated function may also induce apoptotic cell death of the epithelium but may have no physiological correlate. They may, however, have significant application in prevention or control of breast neoplasia.
Animals↗
[Initial results of glutathione-S-transferase GSTM1 and GSTT1 genotypes and genetic predisposition for laryngeal carcinoma].
BACKGROUND: While cigarette smoking and alcohol consumption have been linked to laryngeal carcinoma, the role of genetic factors in determining individual susceptibility is unknown. We describe the role of allelism at glutathione-S-transferase GSTM1, GSTT1, and cytochrome P 450 CYP2D6 loci in determining individual susceptibility to laryngeal squamous cell carcinoma. METHODS: Enzyme genotypes were determined using the polymerase chain reaction in 169 patients suffering T1-T4 laryngeal carcinoma and in 145 controls. RESULTS: While the frequency of the heterozygote GSTM1 A/B genotype was statistically significantly lower in the cases than controls, the frequency of the GSTT1 null genotype was higher in cases than controls. CONCLUSION: These initial data suggest that allelism at GST loci mediates susceptibility to squamous cell carcinoma of the larynx. Thus, GSTM1 A/B appears to be associated with a reduced risk while GSTT1 null confers increased risk. The findings are compatible with the view that genetic predisposition is important in determining risk of this cancer.
Adult↗
Treatment with chemopreventive agents, difluoromethylornithine and retinyl acetate, results in altered mammary extracellular matrix.
The effect of treatment with D-L-2-difluoromethylornithine (DFMO) plus retinyl acetate (RA) on the promotion stage of 1-methyl-1-nitrosourea (MNU)-induced mammary carcinogenesis was evaluated in female Sprague-Dawley rats. Combined treatment was more effective than single agent treatment in decreasing cancer incidence and multiplicity and in prolonging cancer latency. Increased efficacy was associated with reduced morphological complexity of the gland and increased mammary extracellular matrix. Using ovarian hormones to model mitogen stimulation in the mammary gland, DFMO plus RA treatment reduced mammary gland complexity in the absence of an effect on bromodeoxyuridine (BrDU) labeling index measured immunohistochemically. Morphological and biochemical evaluation of these glands revealed increased levels of extracellular matrix in rats treated with chemopreventive agents. Tenascin expression and fibronectin levels were elevated and laminin levels were decreased. The fact that matrix degrading proteinase activity was also increased indicated that tissue remodeling was modulated by these chemopreventive agents. These data provide evidence of alterations in epithelial cell-extracellular matrix interactions that could account in part for the chemopreventive effects of DFMO plus RA.
Animals↗
Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium.
Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.
Journal Article↗
Serum squamous cell carcinoma antigen: a potential marker for benign vulval disease?
Benign vulval disease comprises a variety of disorders and can affect women of all ages. To date, the optimal management of these conditions has been uncertain and not subjected to a systemic prospective approach. It is recognized that benign vulval disease has a potential premalignant potential. Squamous cell carcinoma antigen (SCCA) has shown in different studies to be an effective means of monitoring the course of the disease in cervical carcinoma. Elevated levels of SCCA have been found in the skin. In addition, raised SCCA levels have been found in non-carcinomatous inflammatory dermatoses, and the levels observed correlated with the extent of the disease and the response to therapy. It was thought that SCCA might prove to be a useful marker for benign vulval disease, and in our pilot study the objectives were to determine if levels of SCCA are elevated in patients with that disease and to assess whether there is an association between SCCA and clinical response to treatment.
Antigens, Neoplasm↗
Expression of stromelysin-1 and TIMP-1 in the involuting mammary gland and in early invasive tumors of the mouse.
The mammary gland, during post-lactational involution, is subjected to extensive tissue reconstruction. This process is governed by the concerted expression of extracellular-matrix-degrading enzymes and their inhibitors. During carcinogenesis, the invasive growth of tumor cells is characterized by the penetration of the basement membrane and stromal invasion. We compared the expression of the tissue-remodeling enzymes stromelysin-1, a matrix metalloproteinase, and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), during mammary gland involution and carcinogenesis in mouse. In involuting mammary glands, stromelysin-1 was expressed in myoepithelial cells, whereas TIMP-1 was confined to the stromal tissue. To analyze the involvement of these tissue-remodeling genes in tumor development, we examined mammary tumors of transgenic mice expressing either the activated Ha-ras or c-myc oncogene under the control of a milk-protein gene promoter. In the undifferentiated and metastasizing Ha-ras-induced tumors, stromelysin-1 expression was comparable to that seen in involution, whereas TIMP-1 expression was greatly elevated. During Ha-ras-induced carcinogenesis, stromelysin-1 expression was first detected in the myo-epithelial cells surrounding preneoplastic lesions. In contrast, in the well-differentiated and non-metastatic mammary tumors induced by c-myc, no expression of either gene was observed. Thus, expression of stromelysin-1 and TIMP-1 is confined to the aggressively growing tumors and is induced in the earliest stages of carcinogenesis.
Animals↗
Cloning, expression, and purification of a functional nonacetylated mammalian mitochondrial chaperonin 10.
An intact mouse mitochondrial chaperonin 10 has been cloned, sequenced, and overexpressed in Escherichia coli as a fusion protein harboring an oligohistidine tail at its COOH terminus. The latter was added to simplify protein purification. The purified protein is free of contaminating groES from the bacterial host cells. Edman degradation reveals that the initiator Met residue of the recombinant protein is removed in vivo, similar to the authentic chaperonin 10 purified from rat liver mitochondria. However, in contrast to the latter, the amino-terminal Ala residue of the recombinant protein is not acetylated; the molecular mass determined by electrospray ionization mass spectrometry is 12,350.9 +/- 2.6 daltons, in agreement with that predicted for the nonacetylated protein (12,351.2 daltons). Facilitated protein folding experiments with ribulose-biphosphate carboxylase, under "nonpermissive" in vitro conditions, demonstrate that the recombinant protein is fully functional with groEL. Thus, both the initial rates of protein folding and final yields observed with this heterologous combination are virtually identical to those obtained with groEL and groES. More important, like the authentic protein purified from mitochondria, the recombinant mitochondrial chaperonin 10, but not groES, is functionally compatible with the heptameric chaperonin 60 of mammalian mitochondria.
Acetylation↗
Polymorphism at the glutathione S-transferase GSTM1 locus: a study of the frequencies of the GSTM1 A, B, A/B and null phenotypes in Nigerians.
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Adult↗
Nucleation of the iron core occurs at the three-fold channels of horse spleen apoferritin: an EXAFS study on the native and chemically-modified protein.
Extended X-ray absorbance fine structure measurements have been carried out on the initial Fe(III)-apoferritin complex at a Fe/subunit ratio of 2 in native and modified horse spleen apoferritin. Analysis of the data indicates that in the native protein the iron forms a protein-bound polynuclear cluster (Fe-Fe distance 3.4 A) with a first coordination sphere constituted by 5-6 low-Z atoms, e.g., nitrogen atoms, carboxylate-like ligands or oxo bridges between the iron atoms. Modification of Cys-126, a residue localized on the outer surface of the hydrophilic three-fold channels, with p-chloromercuribenzoate (PMB) or phenylmercuric acetate (PMA) brings about distinctive differences. In particular, in the PMB-reacted protein the feature assigned to the iron-iron interaction disappears from the spectrum, whilst in the PMA-reacted protein the main differences with respect to the native protein are observed at the level of the first coordination sphere. These results confirm the formation of protein-Fe(III)-clusters and localize these sites at the hydrophilic three-fold channels of horse spleen apoferritin.
Animals↗
The mechanism of iron uptake by transferrins: the structure of an 18 kDa NII-domain fragment from duck ovotransferrin at 2.3 A resolution.
The molecular structure of an iron-containing 18 kDa fragment of duck ovotransferrin, obtained by proteolysis of the intact protein, has been elucidated by protein crystallographic techniques at 2.3 A resolution. This structure supports a mechanism of iron uptake in the intact protein whereby the binding of the synergistic (bi)carbonate anion is followed by binding of the metal with the lobe in the open configuration. These stages are then followed by domain closure in which the aspartic acid residue plays a further key role, by forming an interdomain hydrogen-bond interaction in addition to serving as a ligand to the iron. This essential dual role is highlighted by model building studies on the C-terminal lobe of a known human variant. In this variant a mutation of a glycine by an arginine residue enables the aspartic acid to form an ion pair and reduce its effectiveness for both metal binding and domain closure. The X-ray structure of the 18 kDa fragment strongly suggests that the histidine residue present at the iron binding site of the intact protein and arising from the second interdomain connecting strand has been removed during the preparative proteolysis.
Journal Article↗
Plasma protein levels in normal human fetuses: 13 to 41 weeks' gestation.
OBJECTIVES: To establish reference ranges for the levels of alpha-fetoprotein, albumin, prealbumin (transthyretin) alpha-1-antitrypsin, transferrin, ceruloplasmin and total protein in the plasma of normal human fetuses and newborn babies. DESIGN: Prospective study of individual normal cases to fulfil objectives. SETTING: Pathology laboratories of the University of Edinburgh and the biochemistry laboratories of the University of Keele. SUBJECTS: Twenty-two normal fetuses 13 to 22 weeks of gestation and 66 babies born between 24 and 41 weeks gestation. RESULTS: Albumin is the predominant plasma protein throughout gestation. The levels of alpha-fetoprotein and prealbumin fell significantly with increasing gestation, whereas the concentrations of the other proteins studied increased. The ratios of individual proteins to total protein demonstrated similar trends. CONCLUSIONS: This study provides developmental profiles of normal human fetal plasma proteins to serve as possible reference data for abnormal fetuses. Declining levels of prealbumin (transthyretin) were unexpected and suggest a functional role for this protein in early pregnancy.
Blood Proteins↗
Apoptotic cell death and tissue remodelling during mouse mammary gland involution.
During post-lactational mammary gland involution, the bulk of mammary epithelium dies and is reabsorbed. This massive cell death and tissue restructuring was found to be accompanied by a specific pattern of gene expression. Northern blot analysis showed that weaning resulted in a dramatic drop in ODC, a gene involved in synthesis of a component of milk, and the nearly simultaneous induction of SGP-2, a gene associated with apoptotic cell death. These changes were followed by decreases in expression of milk protein genes to basal levels and expression of genes associated with regulation of cell proliferation and differentiation, p53, c-myc and TGF-beta 1. Subsequently, additional genes implicated in stress response, tissue remodelling, and apoptotic cell death were transiently expressed, expression peaking at about 6 days post-weaning. A non-random degradation of DNA yielding the oligonucleosomal length fragmentation pattern typical of apoptotic cell death (Wyllie, 1980; Wyllie et al., 1980) was detected in association with morphological changes and gene expression. The correlations between: (a) changes in morphology, (b) pattern of gene expression and (c) changes in DNA integrity suggest that complementary programs for cell death and tissue remodelling direct post-lactational mammary gland involution.
Animals↗
Glomerular injury induced by hydrogen peroxide: modifying influence of ACE inhibitors.
The sensitivity of isolated glomeruli from normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SHR) strains to oxidant stress was studied by determining the incidence of pyknosis, karyohexis and karyolysis after incubation with different concentrations of hydrogen peroxide (H2O2) (4.7 x 10(-9) - 10(-3) M). Even though the proportion of glomeruli containing nuclei that demonstrated these features increased progressively with increasing concentrations of H2O2, the number of severely damaged glomeruli was relatively small even at concentrations of 4.7 x 10(-3) M. Examination of the surface epithelial cells of glomeruli using scanning electron microscopy revealed no evidence of disturbance of the macroscopic or podocyte structure or, of increased blebbing after H2O2-treatment. These data suggest damage to nuclei is an early result of ROS stress on glomeruli. Preincubation of WKY glomeruli with captopril or lisinopril resulted in a significant drop in the proportion of WKY glomeruli demonstrating structural damage after oxidant stress. In contrast, preincubation of SHR glomeruli with lisinopril had no effect on oxidant-induced changes in the morphology of SHR glomeruli, whereas captopril effected a significant increase in the proportion of glomeruli demonstrating damage at all concentration of H2O2.
Angiotensin-Converting Enzyme Inhibitors↗
Developmental profile of plasma proteins in human fetal cerebrospinal fluid and blood.
Total protein, alphafetoprotein, albumin, prealbumin, alpha-1-antitrypsin, transferrin and ceruloplasmin levels were measured in samples of human fetal and neonatal cerebrospinal fluid (CSF) (97 cases), obtained between 12 and 41 weeks of gestation. In 39 of these cases (13 to 40 weeks gestation) plasma was also available for comparative analysis. CSF was collected from lateral ventricles in the first half of gestation and from the lumbar region in the second. Since these CSF samples were obtained from different sites, the protein levels in the lateral ventricle (early) samples could not be compared directly with those in the lumbar (later) samples. However, the mean protein levels in the lumbar samples were lower than those in the ventricular samples, which is in accord with the decline in CSF protein levels described in maturing animal fetuses. Despite a wide scatter of results, particularly in the first half of gestation, significant decline in the level of CSF alphafetoprotein was demonstrated during both first and second halves of gestation, and of albumin and prealbumin in the second half. No sex differences were found except for ceruloplasmin in lumbar CSF later in gestation, when males had higher levels than females. In fetal plasma, protein levels increased with increasing gestation apart from alphafetoprotein and prealbumin which both declined progressively. CSF/plasma ratios were dissimilar for different proteins, and changed with increasing gestation. These findings support the concept that the human fetal blood brain barrier matures early.
Blood Proteins↗