Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells.

BACKGROUND: LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein. METHODS: Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5. RESULTS: Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network. CONCLUSIONS: Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function.

Induction of endogenous mammary tumor virus expression during hormonal induction of mammary adenoacanthomas and carcinomas of BALB/c female mice.

Treatment of BALB/c female mice with pituitary isografts, under conditions that established mammary hyperplasia in an anovulatory condition, enhanced mammary tumor development with prior onset of dysplastic foci in lobular parenchyma. Tumor onset began at 8 months (mean onset time, 18 mo); the 25-month incidence was 100%. Adenoacanthomatous tumors appeared first. Adenocarcinomas appeared only after more than 14 months of continuous hormone stimulation. Dysplastic and neoplastic changes occurred while blood levels of the three major mammotropic hormones were physiologic. Murine mammary tumor virus (MuMTV) p28 was detected in all tumors tested, independent of time of tumor appearance or tumor type, although keratinizing cells in adenoacanthomatous tumors did not contribute to MuMTV antigen expression. MuMTV gp52 was detected in only a small fraction of cells in a few tumors. MuMTV RNA contents were above normal in all tumors tested. Neither MuMTV structural antigens nor RNA was induced in normal glands by the same hormone treatment that ultimately resulted in dysplasia and tumor formation and elevated levels of these viral markers in neoplasms. The MuMTV RNA in all hormone-induced tumors was readily distinguishable in base sequence from standard MuMTV RNA but indistinguishable from MuMTV RNA recovered from lactating mammary glands of BALB/c females carrying only endogenous MuMTV proviruses, suggesting that endogenous MuMTV RNA sequences were induced in hormone-induced neoplasms. RNA indistinguishable from MuMTV sequences present in hormone-induced primary tumors was also detected in multiple genomic equivalents in two independently derived hormone-induced premalignant alveolar hyperplasias. MuMTV p28 was detected, but gp52 was not. The same hormone stimulus that generated 100% tumors in normal gland greatly accelerated tumor development in premalignant hyperplasias but did not amplify MuMTV RNA or antigens in either hyperplasias or the tumors derived from them. B-type virions were not detected in these tissues, in either hypophyseal implant-stimulated or virgin hosts. Cell-free virions were not detected in culture. These data suggest that the replication of MuMTV induced in hormone-induced neoplasms is defective. Details of its expression suggest that if involved in events leading to tumors, its involvement is insufficient cause for those tumors.

Hormonal induction of mammary tumor viruses and its implications for carcinogenesis.

The purpose of this study was to evaluate the possibility that hormones and mouse mammary tumor virus (MuMTV) are cocarcinogenic for mammary epithelium. Results of our investigations in vivo and in vitro suggest: (a) Hormones promote mammary carcinogenesis in BALB/c females whether MuMTV is germinally (BALB/c) or horizontally (BALB/cfC3H) transmitted; the rate of carcinogenesis in BALB/cfC3H females is substantially faster than it is in BALB/c, but the final mammary carcinoma incidence is approximately the same. The rate-limiting step in malignant transformation in BALB/c, which infectious MuMTV overcomes, is in premalignant transformation from normal. (b) One characteristic of horizontal MuMTV transmission in BALB/c that is not observed in germinal transmission is integration of new MuMTV sequences in mammary cell DNA. Integration is mammary cell specific and constant at 2 to 3 copies/cell from tumor to tumor. (c) MuMTV expression is changed in mammary epithelial cells during hormonal carcinogenesis. The nature of the change is qualitatively similar in both BALB/c and BALB/cfC3H. Expression of envelope glycopeptides (glycoprotein with a molecular weight of 52,000) is induced, which correlates with amplification of MuMTV RNA sequence content. Quantitative differences exist in induced levels in BALB/c and BALB/cfC3H. (d) MuMTV RNA and a glycoprotein with a molecular weight of 52,000 were not inducible with dexamethasone in normal mammary epithelial cells in culture. These structural components were induced in both premalignant (BALB/cfC3H) and malignant (BALB/c and BALB/cfC3H) cells. MuMTV RNA was induced by dexamethasone in normal cells pretreated with 5-iodo-2'-deoxyuridine. (e) Both premalignant and malignant cells have altered (vis-à-vis normal) surfaces, discernible by differences in reactivity with concanavalin A in hemadsorption assays. Indirect evidence suggests that the alteration includes membrane incorporation of MuMTV-related determinants of a glycoprotein with a molecular weight of 52,000. (f) Malignant cells exhibit enhanced sensitivity to insulin for reinitiation of DNA synthesis and mitosis in contact-inhibited homotypical monolayers. These findings have been organized into a hormonal cocarcinogenesis hypothesis in which expression of germinally transmitted MuMTV genes is the proximal cause of neoplastic transformation.

Role of growth factor synthesis in the acquisition of insulin/insulin-like growth factor I independence in rat mammary carcinoma cells.

In previous work, we demonstrated that a subset of carcinogen-induced rat mammary carcinomas consists of cells that are independent of growth factors strictly required by normal rat mammary epithelial (RME) cells for long-term growth in serum-free medium. Furthermore, only those tumors that expressed growth factor independence in vitro were serially transplantable in vivo. The present studies were aimed at determining if the independence of insulin (IN)/insulin-like growth factor I (IGF-I) is mediated by autocrine factors synthesized by the rat mammary tumor (RMT) cells. The results of these experiments indicate that IN/IGF-I-independent RMT cells do not synthesize IGF-I that is detectable at the message or peptide level. Both normal and neoplastic cells do, however, secrete IGF-I-binding activity. Conditioned medium, cell lysates, and cell extracts obtained from growth factor-independent cells do not contain growth factor activity that can substitute for IN for growth of IN-dependent RMT cells. Growth factor-independent cells do not express a density dependence for growth in IN-free medium nor do they respond to exogenous IN or IGF-I in low density growth assays. By contrast, growth factor-dependent cells that were rendered IN/IGF-I independent by transfection with an expression vector containing the IGF-I gene secrete IGF-I-like biological activity that is readily detectable and maintain responsiveness to exogenous IN at low densities. Taken together, these results suggest that growth factor-independent RMT cells are truly autonomous of IN/IGF-I for growth and are not synthesizing a growth factor that satisfies their IN/IGF-I requirement in an autocrine manner.

Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10.

Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 mM) and floating transfers in low calcium (MCF-10F), or by trypsin-Versene passages in the customary (normal) calcium levels, 1.05 mM (MCF-10A). Both sublines have been maintained as separate entities after 2.3 years (849 days) in vitro and at present have been in culture for longer than 4 years. MCF-10 has the characteristics of normal breast epithelium by the following criteria: (a) lack of tumorigenicity in nude mice; (b) three-dimensional growth in collagen; (c) growth in culture that is controlled by hormones and growth factors; (d) lack of anchorage-independent growth; and (e) dome formation in confluent cultures. Cytogenetic analysis prior to immortalization showed normal diploid cells; although later passages showed minimal rearrangement and near-diploidy, the immortal cells were not karyotypically normal. The emergence of an immortal culture in normal calcium media was not an inherent characteristic of the original tissue from which MCF-10 was derived since reactivated cryo-preserved cells from cultures grown for 0.3 and 1.2 years in low calcium were incapable of sustained growth in normal calcium.

Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii.

The patterns of alanine dehydrogenase, glutamate dehydrogenase and malate dehydrogenase activity were studied during the normal vegetative cell cycle and during the processes of gametic differentiation and dedifferentiation in synchronized cultures of Chlamydomonas reinhardtii. During all three phases of growth and differentiation the synthesis of DNA was also measured. During gametic differentiation all three enzyme levels were suppressed compared to vegetative cells although DNA and cell number were comparable. During gametic dedifferentiation no DNA synthesis occurred during the first 24 h cycle and only a doubling during the second. It was not until the third cycle that a normal 4-fold increase in DNA was observed. Cell number followed a similar pattern. Although the levels of alanine dehydrogenase and malate dehydrogenase were uniformly low during the first cycle when glutamate dehydrogenase increased 4-fold, during the second cycle the patterns of these enzymes changed markedly. The enzymes did not attain levels characteristic of vegetative cells until the third cycle.

Heterocyclic amines and genotype of N-acetyltransferases as risk factors for prostate cancer.

A variety of carcinogenic heterocyclic amines are produced during the cooking of meat at high temperatures. These carcinogens are metabolized by N-acetyltransferases (NAT), which are polymorphic in the population. This study examined associations between prostate cancer (PCa) and the consumption of different kinds of meat, heterocyclic amine intake and NAT genotypes. PCa patients and controls were recruited in the Syracuse, NY area. Levels of meat and heterocyclic amine intakes were determined from validated surveys and NAT genotypes were determined by the sequences of PCR-amplified DNA from buccal swabs. A total of 152 cases and 161 controls were eligible for analysis. There was an association between PCa and history of PCa in the first-degree blood relatives (OR = 4.59, 95% CI 2.21-9.70), and family history of bladder cancer (P < 0.02). However, there was no association with the history of other cancers. There was no association between PCa and either 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) intake, or NAT1 and NAT2 genotypes. However, there was a trend of association with MeIQx and with rapid NAT2 and NAT1*10 in combination with PhIP. A new NAT1 allele with a frequency of one out of 544 chromosomes was found in the Caucasian subjects.

PEPMOTIF: a program for locating class I major histocompatibility complex restricted peptides in protein sequences.

PEPMOTIF is a computer program which analyzes protein sequences for the occurrence of peptides up to ten residues in length which contain motifs presented by particular class I major histocompatibility complexes. Any peptide motifs defined by the user can be identified in a protein sequence of interest. PEPMOTIF generates a listing of all motif-containing peptides found in the protein, and two modes of data output are provided: (1) direct printout, or (2) storage in a text file on disk.

Preliminary studies of monoclonal antibody lymphoscintigraphy in malignant melanoma.

Lymphoscintigraphy was performed at 3 and 20 hr following subcutaneous injection of 131I anti-melanoma antibody (Fab) in 11 patients who had surgical resection of lymph nodes (neck, axilla, groin) at 24 hr for suspected metastatic melanoma. Comparable amounts of 125I nonspecific control antibody (Fab) were co-administered. Six patients had nodal metastases and three showed positive images at both time periods. Five patients had no metastases though one was image positive. Four other nondiseased inguinal node groups were image negative. A total of 28 tumored nodes and 110 normal nodes were removed, counted and histologically examined. All metastatic tumors expressed antigen against which the specific Fab was directed. The concentrations of both specific and nonspecific Fab were similar in tumored nodes and both were significantly greater than in normal nodes showed essentially identical intranodal spatial distribution of the specific and control Fab in areas containing tumor. These preliminary results suggest the increased concentration of murine immunoglobulin (Fab) retained in diseased nodes was a nonspecific phenomenon.

A nude rat model for in vivo studies of human transitional cell carcinogenesis.

A xenograft system was developed for studying experimental carcinogenesis of human transitional cells in vivo. Segments of human ureters were tied to an injection port, ligated at the opposite end, implanted into gamma-irradiated nude rats and weekly irrigated through the injection port with fresh PBS solution. Such implants maintained the normal histologic appearance of human urothelium for at least 20 weeks in vivo in the nude rats. In situ hybridization with a human repetitive sequence DNA probe showed that the urothelium and the submucosal connective tissue and smooth muscle were of human origin. The urothelial lining was positive with an immunohistochemical reaction for acidic cytokeratins. This model allows for the long-term direct exposure of human urothelium to bladder carcinogens for the purpose of induction of human transitional cell tumors.

Carcinogenicity of the N-hydroxy derivative of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline and 3, 2'-dimethyl-4-aminobiphenyl in the rat.

Heterocyclic amines and carcinogenic aromatic amines are similarly metabolically activated suggesting that they may have similar organ specificity. Three day-old male ACI/seg rats were injected, i.p., twice a week for 10 weeks with 50 micromol/kg of N-hydroxy-3, 2'-dimethyl-4-aminobiphenyl (N-OH-DMABP; Group II), N-OH-2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx; Group III) or N-OH-2-amino-1-methyl-6-phenylimidaza-[4,5-b]pyridine (N-OH-PhIP; Group IV). Animals in control group (Group I) were similarly injected with solvent alone. The animals were sacrificed at age 68 weeks, and 31, 30, 27 and 31 rats from Groups I, II, III and IV, respectively, were evaluated. Colon carcinomas were found in 0, 15 (P<0.001), 2 and 4 (P<0.06), and bladder transitional cell tumors in zero, two, two and four (P<0.06), in Group I, II, III and IV, respectively. The incidence of atypical hyperplasia of ventral prostate in Groups III and IV, and of anterior prostate and seminal vesicle in all treated groups was also significantly greater (P<0.05). These results suggest that N-OH-PhIP and N-OH-MeIQx may be potential carcinogens for the prostate. Since bladder tumor is rare in ACI rats, N-OH-PhIP may also be a potential carcinogen for the bladder.

Serum and tissue concentrations of cefoxitin and cefotaxime in women undergoing hysterectomy.

In a comparative study of serum and uterine tissue concentrations of cefoxitin and cefotaxime in patients undergoing hysterectomy, 40 patients were randomised to receive either cefoxitin 2g or cefotaxime 2g by intravenous administration. Serum samples were obtained before drug administration, at the ligation of the uterine arteries and at the end of surgery. Cefoxitin, cefotaxime and desacetylcefotaxime concentrations were determined by high performance liquid chromatography. The composite serum half-lives (determined by linear regression) for cefoxitin, cefotaxime and desacetylcefotaxime were 0.8, 0.7 and 2.1 hours, respectively. Although serum concentrations were higher for cefotaxime than for cefoxitin after a 2g dose, the uterine concentrations (at 40 mins) of cefoxitin were higher (51 micrograms/g vs 16 micrograms/g) than those of cefotaxime. After a 2g dose of cefotaxime the desacetylcefotaxime peak uterine concentration was 8 micrograms/g. Both drugs achieved adequate concentrations in serum and uterine tissue to prevent and treat infections caused by common Enterobacteriaceae such as Escherichia coli, with cefotaxime having a longer apparent duration of activity. However, cefoxitin provided serum and uterine concentrations above the minimum inhibitory concentration of Bacteroides fragilis for a longer period than did cefotaxime.

Modification of plasmid and bacteriophage DNA by aromatic amines: effects on survival, template activity, and mutagenicity.

The carcinogenic and mutagenic effects of the aromatic amines are believed to depend on their covalent modification of DNA, primarily through the formation of adducts at C8 of guanine. The actual biologic and biochemical responses to these adducts can be envisioned as the consequence of the abilities of the cell to repair the lesions, with or without fidelity, and the introduction of errors through bypass of the adducts by polymerases. A key question is whether changes in DNA sequence arise through the participation of common repair processes that cause mutations independent of adduct structure. Alternatively, do mutations arise through miscoding during polymerase bypass at the site of the adducts and are, therefore, more likely to produce sequence changes that are more characteristic of adduct structure? This question has been approached using single, site-specific, or randomly introduced aromatic amine DNA adducts in bacterial cells, and in vitro studies with DNA polymerases that employ site-specifically modified templates. The results of both approaches demonstrate that these adducts are distinguished readily by virtue of their structures, thus supporting the conclusion that mutagenic effects of the aromatic amines arise from their structures rather than from their triggering a common inaccurate repair response.

Chemical carcinogenesis of the urinary bladder--a status report.

Cigarette smoking and certain types of occupational exposure to arylamines appear to be the main cause of human urinary bladder cancer. Little is known of the promotion of bladder cancer in humans, although this stage has been demonstrated in rodents. Perhaps as a consequence of initiation, multifactorial alterations of cellular genes occur. These genes include the epidermal growth factor receptor gene, erbB-2, int-2, hst, and H-ras, which exert positive control over cell growth, as well as the suppressor genes Rb-1, and the gene coding for p53. Chromosomal changes such as deletions, translocations and/or amplifications related to these genes may be of significance for prognosis of this disease.

Isolation and characterisation of bacteria from abscesses in the subcutis of cats.

Thirty-six closed abcesses in the subcutis of cats were examined. Of 168 bacterial strains isolated, 121(72%) were anaerobes and 47 (28%) were facultative anaerobes. Twenty-six abscesses contained mixtures of facultative anaerobes and anaerobes, six contained anaerobes only and four contained facultative anaerobes only. Bacteriodes was the genus most commonly isolated (28.6% of all isolates) followed by Fusobacterium (19.0%) and Pasteurella (multocida) (13.1%). Peptostreptococcus anaerobius was the most commonly isolated anaerobic species (13.2% of anaerobic isolates and 9.5% of all isolates)and Past, multocida was the most commonly isolated facultative anaerobe (46.8%; 13.1%of all isolates).

Immunotherapy and gene therapy for renal cell carcinoma.

New immunotherapeutic strategies have significantly improved the management of metastatic renal cell carcinoma, which is otherwise refractory to conventional chemotherapy and radiotherapy. Objective response rates of up to 40% have been achieved in clinical trials using systemic administration of interferon-α, interleukin-2, adoptively-modified lymphokine-activated killer cells, or tumor-infiltrating lymphocytes. With the advent of recombinant genetics, approaches are now available for enhancing host antitumor immunity and improving tumor vaccine. In animal models, tumor vaccines expressing immunostimulatory cytokines have demonstrated the suppression of tumor growth and metastasis, elimination of pre-established tumors, and elicitation of immunity against tumor recurrence. However, most of these vaccines were not beneficial in human. Other approaches with the suppressor gene p53 and herpes simplex virus thymidine synthase gene as a suicide gene system have shown substantial tumor remission and clinical trials are currently underway. Gene therapy with multidrug resistance gene (MDR-1) also is applied for subsequent protection against myelosuppression during high-dose chemotherapy. Moreover, significant treatment improvements have resulted from combinations of gene therapy and immunotherapy along with cytotoxic agents, X irradiation, and biological response modifiers in experimental systems. In general, the future success of cancer gene therapy requires further development of techniques to regulate gene expression and enhancement of antitumor activity and choice of gene with appropriate bioactivity for individual tumors.

Endoscopic third ventriculostomy.

Long-term extracranial shunting for hydrocephalus has numerous drawbacks related to shunt malfunction and infection. In some cases outcome has been very disappointing. We successfully managed 5 patients with acquired aqueductal stenoses with no significant morbidity by the use of an intracranial cerebrospinal fluid diversion, namely a third ventriculostomy. First advocated by Dandy, ventriculostomy was largely passed over in favor of extracranial procedures. With improved surgical techniques, however, ventriculostomy is now considered to be a viable alternative in selected cases. In a further 19 patients, we subsequently broadened our patient selection to include those with Arnold-Chiari malformations, congenital noncommunicating hydrocephalus, and tumors. Two thirds of these children remain without shunts and apart from 1 child developing hemiplegia postoperatively, there has been no significant morbidity. Although the best results have been seen in the late onset groups, even early onset, noncommunicating hydrocephalus has been successfully managed. Even in patients in whom third ventriculostomy has failed and who have subsequently required ventriculoperitoneal shunts, we anticipate that they will remain less dependent on shunts because their hydrocephalus is now communicating, which tends not to have such a rapid onset or extreme levels of raised intracranial pressure.

Ventriculopleural shunts for hydrocephalus: a useful alternative.

From 1969 to 1979, ventriculopleural shunts were inserted in 29 children with progressive hydrocephalus. A standard Pudenz pump with a Raimondi catheter was used for all but 1 child for whom a Holter valve was used. The shunt functioned adequately in 7, but in 18 it had to be changed as a result of symptomatic pleural effusion. From 1979 to 1982, a further series of 52 other patients received ventriculopleural shunts, and these cases have recently been reviewed. The apparatus used was a Portnoy ventricular catheter or a medium or high pressure Heyer Schulte pump with an antisiphon device and a Salmon distal catheter. Three patients developed a shunt infection. One died with a functioning shunt. Four catheters became blocked by adhesions, and in only 1 patient was a peritoneal shunt substituted as a result of symptomatic effusion.

Suprasellar arachnoid cysts: management by cyst wall resection.

Five children with ventricular dilatation (4 boys, 1 girl) had features seen on computer tomographic scan that were consistent with suprasellar arachnoid cysts. All children were investigated with a CT ventriculogram and/or CT cisternogram, and no communication with the cyst was demonstrated. Three children were seen in the 1st year of life and the remaining 2 children were between 1 and 5 years of age. Hydrocephalus and developmental delay were the most common presenting features, followed by visual disturbance, squint, or ataxia. Direct surgical decompression was performed in all 5 patients to avoid long-term placement of a ventriculoperitoneal shunt. A temporary shunt was placed in 2 children because of high intracranial pressure. Direct partial excision of the cyst wall to allow long-term drainage into the basal cisterns or ventricular system was successful in all children. The presence of subdural collections postoperatively required temporary shunting in 2 children. After follow-up for between 10 and 22 months no clinical endocrinological sequelae have been detected, but 2 children have raised serum prolactin levels. Three children are developmentally delayed; one of these has regained some skills since surgery. Direct surgical decompression of suprasellar arachnoid cysts to avoid long-term shunt placement is the preferred method of surgical treatment for this condition.

Third ventriculostomy for shunt infections in children.

Four children with extracranial shunts for noncommunicating hydrocephalus suffered from recurrent or intractable shunt infections. All patients were resistant to or relapsed after treatment with intravenous and intrathecal antibiotics with change of the shunt apparatus. They were treated with neuroendoscopic third ventriculostomy and the removal of all implants, except for a reservoir in one patient. That child later had the reservoir removed because of persistent proteus infection. All patients received antibiotics for approximately 2 weeks after the operation. There was no morbidity associated with the procedure, and all patients remain shunt independent with follow-up periods of 21 to 46 months (mean, 33 mo), although one has needed another third ventriculostomy. We have shown that third ventriculostomy is a successful surgical intervention for the management of shunt infections in patients with noncommunicating hydrocephalus.