A gene on the HER2 amplicon, C35, is an oncogene in breast cancer whose actions are prevented by inhibition of Syk.

BACKGROUND: C35 is a 12 kDa membrane-anchored protein endogenously over-expressed in many invasive breast cancers. C35 (C17orf37) is located on the HER2 amplicon, between HER2 and GRB7. The function of over-expressed C35 in invasive breast cancer is unknown. METHODS: Tissue microarrays containing 122 primary human breast cancer specimens were used to examine the association of C35 with HER2 expression. Cell lines over-expressing C35 were generated and tested for evidence of cell transformation in vitro. RESULTS: In primary breast cancers high levels of C35 mRNA expression were associated with HER2 gene amplification. High levels of C35 protein expression were associated with hallmarks of transformation, such as, colony growth in soft agar, invasion into collagen matrix and formation of large acinar structures in three-dimensional (3D) cell cultures. The transformed phenotype was also associated with characteristics of epithelial to mesenchymal transition, such as adoption of spindle cell morphology and down-regulation of epithelial markers, such as E-cadherin and keratin-8. Furthermore, C35-induced transformation in 3D cell cultures was dependent on Syk kinase, a downstream mediator of signalling from the immunoreceptor tyrosine-based activation motif, which is present in C35. CONCLUSION: C35 functions as an oncogene in breast cancer cell lines. Drug targeting of C35 or Syk kinase might be helpful in treating a subset of patients with HER2-amplified breast cancers.

4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.

BACKGROUND: There is a close correlation between tumour progression and hyaluronan production, either by tumour cells or by stromal cells that are stimulated by tumour-derived factors. Inhibition of tumour stimulation of fibroblast hyaluronan may suppress tumour growth and invasion. OBJECTIVES: To examine the effect of the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) on the growth of and hyaluronan synthesis by fibroblasts and C8161 and MV3 melanoma cell lines, invasion, and inhibition of tumour cell-derived factor activation of fibroblasts. METHODS: Effects of 4-MU on growth and hyaluronan synthesis by fibroblasts and melanoma cells were examined in monolayer culture and fibroblast-contracted collagen lattices, and their effects on the growth and invasion of tumour cells into collagen lattices were also studied. RESULTS: 4-MU caused a dose-dependent growth inhibition of fibroblast and melanoma cells with maximum inhibition at 0·5 mmol L(-1) 4-MU. At this dose, 4-MU inhibited (3) H-glucosamine incorporation into fibroblast glycosaminoglycans by 52%, and hyaluronan synthesis by 64%. The relative inhibition was more pronounced when fibroblasts were stimulated with C8161 melanoma cell-conditioned medium. 4-MU reduced the level of hyaluronan in fibroblast-contracted collagen lattices, and inhibited both the growth on and invasion into the lattices by melanoma cells. This growth inhibition appears to be predominantly independent of inhibition of hyaluronan synthesis. The effect on growth inhibition was reversible, and 4-MU had no effect on apoptosis. CONCLUSIONS: 4-MU is a potent inhibitor of hyaluronan synthesis, induction of stromal hyaluronan accumulation by tumour cells, and fibroblast and melanoma cell proliferation, and results suggest that 4-MU may have potential as a tumour cell anti-invasive and antiproliferative agent.

Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength.

Although a biphasic dependence of cell migration speed on cell-substratum adhesiveness has been predicted theoretically, experimental data directly demonstrating a relationship between these two phenomena have been lacking. To determine whether an optimal strength of cell-substratum adhesive interactions exists for cell migration, we measured quantitatively both the initial attachment strength and migration speed of human smooth muscle cells (HSMCs) on a range of surface concentrations of fibronectin (Fn) and type IV collagen (CnIV). Initial attachment strength was measured in order to characterize short time-scale cell-substratum interactions, which may be representative of dynamic interactions involved in cell migration. The critical fluid shear stress for cell detachment, determined in a radial-flow detachment assay, increased linearly with the surface concentrations of adsorbed Fn and CnIV. The detachment stress required for cells on Fn, 3.6 +/- 0.2 x 10(-3) mu dynes/absorbed molecule, was much greater than that on CnIV, 5.0 +/- 1.4 x 10(-5) mu dynes/absorbed molecule. Time-lapse videomicroscopy of individual cell movement paths showed that the migration behavior of HSMCs on these substrates varied with the absorbed concentration of each matrix protein, exhibiting biphasic dependence. Cell speed reached a maximum at intermediate concentrations of both proteins, with optimal concentrations for migration at 1 x 10(3) molecules/micron2 and 1 x 10(4) molecules/micron2 on Fn and CnIV, respectively. These optimal protein concentrations represent optimal initial attachment strengths corresponding to detachment shear stresses of 3.8 mu dyne/micron2 on Fn and 1.5 mu dyne/micron2 on CnIV. Thus, while the optimal absorbed protein concentrations for migration on Fn and CnIV differed by an order of magnitude, the optimal initial attachment strengths for migration on these two proteins were very similar. Further, the same minimum strength of initial attachment, corresponding to a detachment shear stress of approximately 1 mu dyne/micron2, was required for movement on either protein. These results suggest that initial cell-substratum attachment strength is a central variable governing cell migration speed, able to correlate observations of motility on substrata differing in adhesiveness. They also demonstrate that migration speed depends in biphasic manner on attachment strength, with maximal migration at an intermediate level of cell-substratum adhesiveness.

Composite Membranes: The Permeation of Gases through Deposited Monolayers.

We have measured permeabilities of deposited monolayers of stearic acid varying in thickness from 8 to 48 deposited layers. These results are compared with permeabilities of polymeric films and insoluble monolayers. The technique may have applicability in the fabrication of synthetic membranes.

Calibrated membranes with coated pore walls.

Extremely uniform small-radius pores, formed in thin mica membranes, have been coated with monoand multimolecular layers of fatty acids. Stearate monolayers orient themselves on the pore walls in layers 25 to 28 angstroms thick. The resulting membranes, with radii of the order 100 angstroms or less, may have applications in simulating certain features of biological membranes and in fabricating highly selective membranes for differential dialysis or ultrafiltration.

Bubble formation in physical and biological systems: a manifestation of counterdiffusion in composite media.

The counterdiffusion of gases across a composite layer can lead to supersaturation and development of bubbles within the layer. A physicochemical model has been derived to predict the extent of such supersaturation; experiments with inert liquid layers confirm predictions. These findings explain the evolution of cutaneous lesions observed in man during simulated deep-sea dives and the cutaneous lesions and intravascular bubbles experimentally induced in pigs by exchanging certain inert gases across the skin. The phenomena associated with counterdiffusion have widespread physical and biological implications.

Phase I trial of carmustine plus O6-benzylguanine for patients with recurrent or progressive malignant glioma.

PURPOSE: The major mechanism of resistance to alkylnitrosourea therapy involves the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), which removes chloroethylation or methylation damage from the O(6) position of guanine. O(6)-benzylguanine (O(6)-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial of carmustine (BCNU) plus O(6)-BG to define the toxicity and maximum-tolerated dose (MTD) of BCNU in conjunction with the preadministration of O(6)-BG with recurrent or progressive malignant glioma. PATIENTS AND METHODS: Patients were treated with O(6)-BG at a dose of 100 mg/m(2) followed 1 hour later by BCNU. Cohorts of three to six patients were treated with escalating doses of BCNU, and patients were observed for at least 6 weeks before being considered assessable for toxicity. Plasma samples were collected and analyzed for O(6)-BG, 8-oxo-O(6)-BG, and 8-oxoguanine concentration. RESULTS: Twenty-three patients were treated (22 with glioblastoma multiforme and one with anaplastic astrocytoma). Four dose levels of BCNU (13.5, 27, 40, and 55 mg/m(2)) were evaluated, with the highest dose level being complicated by grade 3 or 4 thrombocytopenia and neutropenia. O(6)-BG rapidly disappeared from plasma (elimination half-life = 0. 54 +/- 0.14 hours) and was converted to a longer-lived metabolite, 8-oxo-O(6)-BG (elimination half-life = 5.6 +/- 2.7 hours) and further to 8-oxoguanine. There was no detectable O(6)-BG 5 hours after the start of the O(6)-BG infusion; however, 8-oxo-O(6)-BG and 8-oxoguanine concentrations were detected 25 hours after O(6)-BG infusion. The mean area under the concentration-time curve (AUC) of 8-oxo-O(6)-BG was 17.5 times greater than the mean AUC for O(6)-BG. CONCLUSION: These results indicate that the MTD of BCNU when given in combination with O(6)-BG at a dose of 100 mg/m(2) is 40 mg/m(2) administered at 6-week intervals. This study provides the foundation for a phase II trial of O(6)-BG plus BCNU in nitrosourea-resistant malignant glioma.

Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF.

Estrogen rapidly activates the mitogen-activated protein kinases, Erk-1 and Erk-2, via an as yet unknown mechanism. Here, evidence is provided that estrogen-induced Erk-1/-2 activation occurs independently of known estrogen receptors, but requires the expression of the G protein-coupled receptor homolog, GPR30. We show that 17beta-estradiol activates Erk-1/-2 not only in MCF-7 cells, which express both estrogen receptor alpha (ER alpha) and ER beta, but also in SKBR3 breast cancer cells, which fail to express either receptor. Immunoblot analysis using GPR30 peptide antibodies showed that this estrogen response was associated with the presence of GPR30 protein in these cells. MDA-MB-231 breast cancer cells (ER alpha-, ER beta+) are GPR30 deficient and insensitive to Erk-1/-2 activation by 17beta-estradiol. Transfection of MDA-MB-231 cells with a GPR30 complementary DNA resulted in overexpression of GPR30 protein and conversion to an estrogen-responsive phenotype. In addition, GPR30-dependent Erk-1/-2 activation was triggered by ER antagonists, including ICI 182,780, yet not by 17alpha-estradiol or progesterone. Consistent with acting through a G protein-coupled receptor, estradiol signaling to Erk-1/-2 occurred via a Gbetagamma-dependent, pertussis toxin-sensitive pathway that required Src-related tyrosine kinase activity and tyrosine phosphorylation of tyrosine 317 of the Shc adapter protein. Reinforcing this idea, estradiol signaling to Erk-1/-2 was dependent upon trans-activation of the epidermal growth factor (EGF) receptor via release of heparan-bound EGF (HB-EGF). Estradiol signaling to Erk-1/-2 could be blocked by: 1) inhibiting EGF-receptor tyrosine kinase activity, 2) neutralizing HB-EGF with antibodies, or 3) down-modulating HB-EGF from the cell surface with the diphtheria toxin mutant, CRM-197. Our data imply that ER-negative breast tumors that continue to express GPR30 may use estrogen to drive growth factor-dependent cellular responses.

Analysis of the pobA and pobR genes controlling expression of p-hydroxybenzoate hydroxylase in Azotobacter chroococcum.

We report the cloning and analysis of a gene and its cognate regulatory element from a member of the Azotobacteriaceae which are involved in the breakdown of an aromatic compound. The genes from Azotobacter chroococcum encoding p-hydroxybenzoate hydroxylase (pobA) and its regulatory protein (pobR) were cloned from a genomic library and sequenced. Sequence analysis of pobA revealed homology with other bacterial p-hydroxybenzoate hydroxylase enzymes. Residues essential to the structure and function of the enzyme have been conserved. The pobR gene encodes a DNA binding regulatory protein with similarity to proteins from the AraC/XylS family of transcriptional activators. A fragment containing both pobA and pobR was cloned into pUC19 and p-hydroxybenzoate hydroxylase activity was induced in Escherichia coli by the addition of p-hydroxybenzoate. A frame-shift mutation introduced into the pobR gene prevented expression of p-hydroxybenzoate hydroxylase, indicating that PobR is the protein required for transcription of pobA. Interestingly, A. chroococcum PobR has no homology to the PobR protein that is the transcriptional activator of pobA in Acinetobacter strain ADP1, a protein that is homologous to the IclR family of transcriptional regulators. However, PobR from A. chroococcum is homologous to several other proteins, suggesting that these proteins will also function as transcriptional activators of pobA.

Phase I study of Gliadel wafers plus temozolomide in adults with recurrent supratentorial high-grade gliomas.

Both Gliadel wafers [1,3-bis(2-chloroethyl)-1-nitrosourea] and temozolomide (TEMO) have been shown in independent studies to prolong survival of patients with recurrent malignant glioma following surgery and radiotherapy. On the basis of preclinical evidence of synergism between Gliadel wafers and TEMO, a phase I study was designed to evaluate the toxicity of combining these 2 agents in the treatment of patients with recurrent supratentorial malignant glioma. All patients had surgical resection of the tumor at relapse, and up to 8 Gliadel (3.85%) wafers were placed in the surgical cavity following resection. Two weeks after surgery, TEMO was given orally daily for 5 days. Cohorts of 3 patients received TEMO at daily doses of 100 mg/m2, 150 mg/m2, and 200 mg/m2, respectively. Patients were assessed for toxicity 4 weeks after start of the first course of TEMO. Contrast-enhanced MRI of the brain was used to assesstumor response after the first cycle of TEMO. Patients with stable disease or response after the first cycle of TEMO were allowed to continue treatment at the same dose every 4 weeks for 12 cycles or until disease progression or unacceptable toxicity. Ten patients with a median age of 47 years (range, 22-66 years) were enrolled in this study. There were 7 patients with glioblastoma multiforme and 3 patients with anaplastic astrocytoma. Three patients were treated with TEMO at the first dose level of 100 mg/m2, 4 at the second dose level of 150 mg/m2, and 3 at the third dose level of 200 mg/m2. The 10 patients received a median of 3 cycles (range, 1-12 cycles) of TEMO following placement of Gliadel wafers. The treatment was well tolerated, with only 1 patient suffering grade III thrombocytopenia at the highest dose level. Two patients at each dose level had no evidence of disease progression after treatment. Four patients suffered progressive disease on therapy. Our study demonstrates that TEMO can be given safely after placement of Gliadel (3.85%) wafers. The recommended dosage for TEMO for a phase II study of this combination is 200 mg/m2 per day for 5 days.

Neurologic emergencies in the cancer patient.

Neurologic complications of cancer and its therapy are varied and common, but there are few true neurologic emergencies. However, when a neurologic emergency does occur, rapid diagnosis and treatment can preserve neurologic function and, in some circumstances, save a life. Epidural spinal cord compression, raised intracranial pressure (ICP), status epilepticus, and intracerebral hemorrhage (ICH) are the most common neurologic emergencies in the cancer patient. This chapter details the clinical features, possible etiologies, diagnostic tests, and treatment options for each of these complications.

Gas flux through human skin: effect of temperature, stripping, and inspired tension.

The flux of He and O2 through intact adult human skin was measured at various inspired concentrations and skin temperatures. The skin surface was then stripped with cellophane tape to alter the diffusional conductance of the stratum corneum. He flux for stripped skin was used to estimate skin perfusion as a function of local temperature, and diffusional conductance for O2 was estimated from O2 flux and perfusion. The flux of He or O2 at constant skin temperature can be related to inspired concentration by a simple linear model. Increasing surface temperature in the range 33-43 degrees C produced a much larger increase in O2 flux than in He flux for intact skin. Skin stripping greatly increased skin O2 flux. Estimated skin conductance for O2 showed a more marked temperature dependence than estimated skin perfusion. The results suggest that raising skin temperature in the range 38-43 degrees C has only a modest effect on skin perfusion and that stratum corneum conductance may have a major role in the large increase of O2 flux with temperature.

Permeation of inert gases through human skin: modeling the effect of skin blood flow.

We present an analytic method for determining the effects of skin perfusion--vasculature and flow rates--on the flux of inert gases through human skin. We systematically specify the underlying blood flow and examine the resulting fluxes of several gases, allowing for the appropriate tissue resistances. For physiological flows, the stratum corneum has an effect equivalent to a series resistance. Helium flux at low total flow depends primarily on subdermal perfusion, but at higher flow, middermal and subpapillary effects become important. The fluxes of less permeable gases, such as argon and xenon, depend on middermal and subpapillary flow at lower total flows. From any single measurement of gas flux, it is difficult to establish an unambiguous value for the underlying blood flow, but the simultaneous measurement of different gases narrows the range of plausible conditions.

What about the other breast? A review of a series of bilateral breast carcinomas.

The BreastScreen Queensland Brisbane Southside BreastScreen Service reports on a study of 10 cases of bilateral breast carcinomas from a total cancer population of 217 cases. All cases were patients of screening examinations that were recalled for a suspicious lesion in one breast. Two cases were mammographically suspicious of bilateral tumours. In eight cases, tumours were ultrasonically visible in both breasts and in two further cases, the suspicion of bilateral malignancy was raised by the presence of bilateral microcalcification. It is not the purpose of this paper to provide a statistical analysis of the occurrence of bilateral breast cancer. This is a radiological paper from a breast screening service reporting on findings that conventional wisdom may find unusual. The incidence of bilateral breast malignancy in the study was found to be somewhat higher than expected. These cases have been diagnosed by the utilization of a particularly high standard of ultrasound and mammography, performed and interpreted by diagnosticians possessing an elevated level of suspicion of the possible presence of a second primary lesion. It is therefore proposed that an increased rate of diagnosis of bilateral tumours is possible with an evolution of assessment protocols, combined with quality ultrasound and mammography.

Amphicarpum purshii and the "pessimistic strategy" in amphicarpic annuals with subterranean fruit.

Amphicarpum purshii Kunth is an annual amphicarpic grass of recently disturbed sandy areas in the Coastal Plain of eastern North America, producing small aerial and larger subterranean seeds. At study sites on the eastern edge of the Pine Barrens of New Jersey, the temporal aspects of reproductive allocation were investigated through biweekly whole-plant field harvests of 25 plants each throughout the 1980 growing season, and the survivorship of plants arising from aerial and subterranean seeds was monitored in a total of 10 high and 10 low density quadrats at two sites differeing in soil moisture. Plants arising from subterranean propagules began to allocate early (July 16) considerable energy to subterranean reproduction (ca 40% of whole-plant dry weight by the last half of August); allocation to aerial culms and flowers did not begin until mid-August and never exceeded 3% on a population basis. Plants arising from aerial propagules ("aerial plants") produced only subterranean flowers, and these flowers first appeared 2 wk later than they did on "subterranean plants." Survivorship was greater for the subterranean seedlings at both the dry and wet sites and at low and high densities, and aerial plants showed significantly less total growth and seed production than subterranean plants. The "pessimistic strategy" (early allocation of energy to large subterranean propagules) in the annual Amphicarpum has its selective basis in the relative vigor, survival, and timing and amount of reproduction of the two types of seedlings, and appears comparable to the allocation strategies of at least eight other amphicarpic annuals in at least five other families of plants.

The role of seed depth, litter, and fire in the seedling establishment of amphicarpic peanutgrass (Amphicarpum purshii).

Amphicarpum purshii is an annual grass which mostly grows in disturbed areas of the New Jersey Pine Barrens, USA. It is amphicarpic, producing spikelets (and seeds) both above and below the soil surface. Previous research has shown that subterranean seed production ensures reproduction in the event of a major disturbance such as fire and results in rapid post-burn colonization of these sandy habitats. The effects of fire, litter, and seed depth were further examined by planting subterranean seeds at four depths in 16 litter-covered flats buried at ground level and comparing plants arising from burned flats with those in undisturbed litter-covered flats. At 0 and 1 cm depth, rates of seedling emergence were lowest in burned flats. Surface-sown seeds produced seedlings more likely to desiccate. Sowing depth had a greater influence on most measured characters than burning treatments. The mean depth of subterranean seed placement by Amphicarpum is 3.5 cm and this coincides with the seed depth from which plants showed the greatest height growth, shoot biomass, and reproductive output. In a second experiment, subterranean seeds on the bare soil surface in clay pots were more likely to lose viability and less likely to germinate than seeds protected by litter or burial in soil. In addition to providing protection from fire, placement of seeds below ground in the sandy habitat of peanutgrass provides conditions more suitable for seed survival and subsequent seedling establishment.

The shift in aerial/subterranean fruit ratio in Amphicarpum purshii: causes and significance.

Amphicarpum purshii Kunth, an annual grass, produces both small aerial and larger subterranean seeds, and previous research has indicated that the ratio of the number of viable aerial seeds to the number of viable subterranean seeds decreases with secondary succession and/or the lack of frequent disturbance. The objective of this research was to determine if increasing density and/or greater seed depth could produce this shift in reproductive allocation. Plants arising from aerial and subterranean seeds were grown in pure and mixed cultures at varying densities in a greenhouse to note the effects of intraspecific competition on biomass allocation. In addition, subterranean seeds were sown at varying depths to note the effects of seed depth. Results showed that the growth of plants from aerial seeds was severely depressed in mixtures of plants from the two types of seeds. Increasing density in both pure and mixed cultures led to drastic decreases in allocation to aerial seeds, but the percentage allocation to subterranean seeds was not significantly reduced. Greater seed depth led to decreased emergence rates and increased percentage allocation to subterrancean seeds. It is suggested that as secondary succession progresses, A. purshii and other plants increase in abundance, and the increasing density and the deeper burial of subterrranean seeds result in plants producing mostly subterranean seeds which accumulate in the soil seed bank. These strongly indurate propagules remain viable following the disappearance of Amphicarpum plants in secondary succession and can give rise to "instant populations" upon subsequent vegetation removal and/or soil disturbance.

High mortality of domestic turkeys associated with Highlands J virus and eastern equine encephalitis virus infections.

High mortality occurred in two flocks of commercial turkey hens placed in southern North Carolina in fall 1991. Daily mortality peaked at 3.19% in Flock 1 and 3.79% in Flock 2. Clinical signs included restlessness, somnolence, vocalization, and acute death. Gross lesions included atrophy of the bursa of Fabricius, thymus, and spleen, and watery intestinal contents. Microscopic changes included moderate to marked lymphocyte necrosis and depletion in the bursa, thymus, and spleen, widely scattered necrosis of pancreatic acinar cells, and mild villous atrophy and fusion in the jejunum and ileum with cuboidal to low columnar epithelial cells covering the villous tips. In Flock 1, at 27 days of age, reovirus and picornavirus particles were detected in the feces. One week later, togavirus-like particles were observed in fecal contents, and two of seven serum samples showed seroconversion to Highlands J virus. Eleven days later, five of six serum samples were positive for antibodies against Highlands J virus, with a fourfold increase in the geometric mean titer. In Flock 2, seroconversion to eastern equine encephalitis virus was observed in four of 10 serum samples 11 days after the onset of clinical signs. Based on the above observations, it is suspected that these alphaviruses were the cause of the clinical syndrome.

Skeletal lesions associated with a naturally occurring poult enteritis.

One-day-old poults were placed on contaminated litter on which poults previously had developed an enteric disease characterized by diarrhea, increased mortality, and stunting. These exposed birds were examined for clinical signs and pathologic changes in bone and parathyroid glands compared with controls. Intestinal and fecal samples were examined for potential pathogens. Exposed poults varied in size as early as day 8 and had significantly decreased weight gains and reduced shank lengths on days 8, 12, 16, and 21. The proximal tibial growth plate was narrowed. The mineralized hypertrophy zone was decreased in length and contained multiple non-mineralized bands on days 8, 12, 16, and 21. Metaphyseal trabeculae were reduced in amount on days 16 and 21. Parathyroid glands were hyperplastic on days 16 and 21. The bone and parathyroid gland lesions indicated that mineral homeostasis was being maintained at the expense of the skeleton during the enteric disease. A specific etiology for the enteric disease was not determined. Cryptosporidium, rotavirus, paramyxovirus, and Salmonella were identified in the exposed poults, and paramyxovirus and Salmonella were identified in the controls.

Association of amoebae and actinomyces in an intrauterine contraceptive device user.

A patient wearing an intrauterine contraceptive device (IUD) was diagnosed as being colonized by Actinomyces and amoebae by examination of a routine Papanicolaou smear. The patient received vaginal treatment with metronidazole, which temporarily eliminated the protozoa; however, it was necessary to remove the IUD to definitively eradicate the microorganisms. Follow-up Papanicolaou smears taken after removal of the IUD have been normal and free of both microorganisms.