Disrupting Na⁺, HCO3⁻-cotransporter NBCn1 (Slc4a7) delays murine breast cancer development.

Increased metabolism and insufficient blood supply cause acidic waste product accumulation in solid cancers. During carcinogenesis, cellular acid extrusion is upregulated but the underlying molecular mechanisms and their consequences for cancer growth and progression have not been established. Genome-wide association studies have indicated a possible link between the Na⁺, HCO3⁻-cotransporter NBCn1 (SLC4A7) and breast cancer. We tested the functional consequences of NBCn1 knockout (KO) for breast cancer development. NBCn1 protein expression increased 2.5-fold during breast carcinogenesis and was responsible for the increased net acid extrusion and alkaline intracellular pH of breast cancer compared with normal breast tissue. Genetic disruption of NBCn1 delayed breast cancer development: tumor latency was ~50% increased while tumor growth rate was ~65% reduced in NBCn1 KO compared with wild-type (WT) mice. Breast cancer histopathology in NBCn1 KO mice differed from that in WT mice and included less aggressive tumor types. The extracellular tumor microenvironment in NBCn1 KO mice contained higher concentrations of glucose and lower concentrations of lactate than that in WT mice. Independently of NBCn1 genotype, the cleaved fraction of poly(ADP-ribose) polymerase (PARP)-1 and expression of monocarboxylate transporter (MCT)1 increased while phosphorylation of Akt and ERK1 decreased as functions of tumor volume. Cell proliferation, evaluated from Ki-67 and phospho-histone H3staining, was ~60% lower in breast cancer of NBCn1 KO than that of WT mice when corrected for variations in tumor size. We conclude that NBCn1 facilitates acid extrusion from breast cancer tissue, maintains the alkaline intracellular environment and promotes aggressive cancer development and growth.

Growth and morphological transformations of Helicobacter pylori in broth media.

Helicobacter pylori, a cause of peptic ulcer disease and certain types of gastric cancers, has usually been cultured on diverse agar-based media, resulting in a requirement for 2 to 4 days of growth at 37 degrees C. We have developed a novel broth medium consisting of a base medium supplemented with 2% newborn calf serum, Mg2+, Cu2+, Fe2+, Zn2+, Mn2+, and 1 mg of lysed human erythrocytes per ml. This medium supports rapid growth of H. pylori, with a doubling time of about 50 min. Optimal growth was obtained in a pH range higher than that supporting most other gram-negative bacteria (at pH 8.5). H. pylori cultured in this supplemented broth retains the spiral morphology seen in both histological sections and cultures from agar-based media and also retains a high urease activity. After 18 h in this broth, H. pylori transforms to a coccal form with a complete loss of urease activity. Previously these cocci have been reported to be senescent, since they could not be subcultured on agar medium. Our experiments suggest that some of the cocci can revert back to the spiral morphology with full recovery of urease activity when subcultured in fresh microaerobic broth medium.

Cloning and sequencing of parafusin, a calcium-dependent exocytosis-related phosphoglycoprotein.

A cDNA for parafusin, an evolutionarily conserved phosphoglycoprotein involved in exocytosis, has been cloned and sequenced from a unicellular eukaryote, Paramecium tetraurelia. A Paramecium cDNA library was screened with an oligonucleotide probe synthesized to an internal amino acid sequence of isolated parafusin. The insert was 3 kb long with an open reading frame of 1.75 kb. Data base searches of the deduced amino acid sequence showed that Paramecium parafusin had a 50.7% sequence identity to rabbit muscle phosphoglucomutase, although no detectable phosphoglucomutase activity has been detected in isolated parafusin. The deduced parafusin amino acid sequence had four inserts and two deletions, which might confer on the protein specific functions in signal transduction events related to exocytosis. Furthermore, searches for potential phosphorylation sites showed the presence of a protein kinase C site (KDFSFR) specific to parafusin. Southern blot analysis with probes specific for parafusin and phosphoglucomutase suggested that these proteins were products of different genes. We propose that parafusin and phosphoglucomutase are members of a superfamily that conserve homologies important for the tertiary structure of the molecules.

The activity of parafusin is distinct from that of phosphoglucomutase in the unicellular eukaryote Paramecium.

In this paper we identified the presence of a Paramecium phosphoglucomutase enzymatic activity which is clearly distinct from that of parafusin-the exocytosis-related phosphoglycoprotein. Since the recently cloned parafusin showed homology to rabbit muscle phosphoglucomutase, we have designed a specific peptide parafusin antibody-generated to a region not present in any known sequenced phosphoglucomutases-to distinguish parafusin from the Paramecium phosphoglucomutase. Separation of these two proteins was obtained using liquid chromatography, enzymatic activity assay and immunoblotting analysis with the specific parafusin peptide antibody. Parafusin fractions incorporated [B35S]UDP-Glc but not [35S]Glc-1-P whereas Paramecium phosphoglucomutase fractions incorporated [35S]Glc-1-P but not [B35S]UDP-Glc. This indicates that these two proteins are separate entities exhibiting different properties and most likely distinct functions in the cell.

Characteristics of dividing and non-dividing Tetrahymena cells at different physiological states.

Eight defined physiological states of Tetrahymena pyriformis are described. For dividing cells the states comprise: 1. Exponentially growing cells, 2. Cells at late exponential growth phase, 3. Cells kept at a high cell concentration, 4. Cells shifted up or down by change of medium, temperature or degree of aeration. For non-dividing cells the states are: 5. Cells at stationary phase, 6. Cells during starvation, 7. Cells during shift-up after long-term starvation, 8. Cells at self-induced hypoxia. The different cellular states are described by one or more of the following characteristics: growth rate, volume, swimming speed, oxygen consumption and by the oxygen saturation and the pH in the medium. The results show that T. pyriformis grows equally well in proteose-peptone (PY) medium from 1 cell ml(-1) to 10(3) cells ml(-1) as from - e.g. - 10(2) to 10(5) cells ml(-1). The maximum cell concentration obtained depends on the medium and the availability of oxygen. At shift-down by decrease of temperature the cells grow slower and obtain a considerable oversize. Single cells tolerate starvation for 12 days. The cell volume (electronically determined) decreased from about 7000 µm(3) to about 200 µm(3). Long-term starved cells may be upshifted. Thereby growth without cell division can be studied until the cell volume approaches 2100 µm(3) which is the minimum volume of division competence. Under certain conditions cells may grow into self-induced hypoxia leading to growth arrest. These cells will attain an oversize. The swimming speed at 28°C of exponentially growing cells is 0.33 to 0.59 mm sec(-1) depending on the medium. At lower temperature the swimming speed is decreased. In PY-medium the values are: 28°C (0.57), 16°C (0.50), 9°C (0.37). During starvation the swimming speed decreases from about 0.6 to about 0.1 mm sec(-1) (after 6 days). The oxygen consumption is for state 1 cells: 3.9 µl O(2)/10(6) cells min(-1) (maximal value). The value of hypoxic cultures is 2.1, for cells kept at high concentration 0.4, and for starved cells (24 h) 0.2.

Division competence in Tetrahymena: determination of minimum cell volume and rate of nutrient uptake.

Cell volume and doubling time have been determined for exponentially growing Tetrahymena pyriformis cells in broth medium with and without glucose and in media made from these media by dilution with water. The cells tolerate media with dry weights from 105 down to 0.06 g/L. In the diluted media the cells have small volumes and the doubling time is increased. When the cell volume increase per time per cell in a given medium is expressed as a function of the cell volume in this same medium, a direct proportionality is found. From this equation the minimum cell volume of division competence (MVDC) can be found. It is 2,100 microns 3 for T. pyriformis at 28 degrees C. The lag period resulting from an upshift of exponentially growing cells from diluted media to more concentrated media is a function of the initial and resulting cell volumes and MVDC. The increase in cell volume per unit of time for a given cell depends on the dry weight of the medium. This parameter can be transformed to mass increase per cell surface area per time, which represents rate of nutrient uptake. When plotted against the dry weight of the media, a Michaelis-Menten-like curve is obtained with two Km values of 3.8 and 0.08 g/L with corresponding Vmax values of 20 and 4 ng/cm2.s. The low Km value (0.08 g/L) indicates that Tetrahymena is able to take up nutrients from highly diluted media. The high value of Vmax (20 ng/cm2.s) increases the ability of growth in more concentrated media.(ABSTRACT TRUNCATED AT 250 WORDS)

Misonidazole combined with split-course radiotherapy in the treatment of invasive carcinoma of larynx and pharynx: report from the DAHANCA 2 study.

Between October 1979 and May 1985, 626 patients with pharynx and larynx carcinoma were randomized to two different split-course radiation regimens and given either misonidazole (MISO; 11 g/m2) or placebo during the initial 4 weeks of treatment. Patients in the different treatment groups were evenly distributed among stages. The small number of females (136) gave an uneven distribution, thus making analysis difficult. The results show that females had a statistically better loco-regional control (45 vs. 33%; 5-year actuarial value). Overall, the MISO treated group did not have a significantly better control rate than the placebo groups (37 vs. 34%). However, a difference was found in patients with pharynx carcinomas (38 vs. 27%; p less than 0.05). The pre-irradiation hemoglobin (Hb) concentration was found to be a prognostic parameter. In females, loco-regional control for IIb values below or above 8 mmol/l were 37 and 47%, respectively. In males, the same values were below or above 9 mmol/l 26 and 38%. Hb influence on local control was only seen in supraglottic and pharynx tumors. This effect was independent of tumor size, which also was of prognostic value. In addition Hb concentration and MISO had an apparent additive effect. Thus in the male pharynx group, placebo patients with low Hb had a 14% disease rate compared to 40% in MISO treated patients with Hb above 9 mmol/l. MISO induced significant peripheral neuropathy in 26% of the treated patients, whereas other drug related side effects were minimal and tolerable.

Cell volume and dry weight of cultured Tetrahymena.

Cultures of Tetrahymena pyriformis, T. hegewishi and T. malaccensis have been studied with regard to control of cell volume and cellular dry weight. Cell volume was measured on cells suspended in 0.9% sodium chloride + 0.1% sodium azide using a Multisizer cell counter (Coulter). Tetrahymena were grown at different temperatures and under various up- or downshift conditions. In all cases the changes in cell volume are paralleled by changes in cellular dry weight. The volume and the dry weight of a Tetrahymena cell are determined by the particular medium and the growth temperature. Large cells are seen in concentrated media and at low growth temperatures resulting in cell volumes up to 17,000 microns 3, whereas starving cells decrease gradually towards 700 microns 3 or even smaller sizes. It is proposed that lag phase observed at up- and downshift is to a large extent due to the necessary adjustment of the cell volume to the new conditions.

Thrombosis caused by polyurethane double-lumen subclavian superior vena cava catheter and hemodialysis.

During an 18-month period, 82 consecutive patients (37 women and 45 men), with a mean age of 50 yr (range 15 to 74), underwent hemodialysis with 91 polyurethane double-lumen subclavian superior vena cava catheters inserted via the right subclavian vein. Upon catheter removal, venograms were performed in 53 which revealed thrombosis in 13. Autopsy performed in 13 other patients revealed thrombosis in three. Mean catheter duration was 27 days (range 1 to 138). Catheter duration was significantly (p less than .01) longer in the 16 cases complicated by thrombosis. There was no significant difference in the frequency of thrombosis between men and women. Two patients developed symptoms of subclavian vein occlusion after the creation of an arteriovenous fistula on the same side as a previous hemodialysis catheter. Overall, thrombosis was found in 23% and increased with longer catheter duration. We recommend phlebography to exclude thrombosis before creating an arteriovenous fistula on the same side as the previous hemodialysis catheter. There was no case of clinical discernible pulmonary embolism.

Comparison of conventional and split-course radiotherapy as primary treatment in carcinoma of the larynx.

Based on our experience with conventional, daily irradiation, a split-course radiation schedule was introduced in 1978. The schedule, which was based on Cohen's models for squamous cell carcinoma and vascular damage respectively, predicted an improved tumour control and a reduced rate of late complications, e.g. late oedema, if the conventional, daily treatment was replaced by a split-course schedule. The schedule has later been abandoned, but the experience gained from split-course treatment at various dose levels has been analysed and the results compared with those obtained by conventional radiation. The data allowed construction of dose-response curves and estimation of iso-effect doses. Split-course treatment was associated with a significantly reduced therapeutic ratio because, disappointingly, it did not improve tumour control, and the severity of late complications grew. No late complications were avoided by introducing a 3-week pause in the radiation therapy regimen, nor was the tumour response improved despite a 12-Gy increase in total dose. This indicates a significant repopulation corresponding to more than 0.5 Gy/day, equivalent to an up to 100-fold increase of the number of clonogenic tumour cells during the pause--an increase that occurred despite the decrease, clinically, of the tumours during this period.

An investigation of new commercial samples of methyl green and pyronin Y.

New commercial samples of Methyl Green (Gurr Certistain), Pyronine G (Gurr Certistain) and Pyronin Y (Polysciences) have been investigated using spectrophotometry, thin layer chromatography and nuclear magnetic resonance, in addition to standardized simultaneous and sequential staining methods using purified Ethyl Green and pure Pyronin Y as reference dyes. The Methyl Green was found to be Ethyl Green contaminated with Crystal Violet. It did not have any advantages compared with Ethyl Green supplied by American dye companies. The Pyronine G sample was Pyronin Y with a high dye content that gave good staining results when used with purified Ethyl Green. Pyronin Y from Polysciences was found to be essentially pure Pyronin Y.

Obstructive sleep apnea initiated by a lax epiglottis. A contraindication for continuous positive airway pressure.

Treatment with nasal continuous positive airway pressure (CPAP) was attempted in a patient with severe obstructive sleep apnea. However, application of nasal CPAP gave the patient a feeling of being suffocated. This was later documented by cine-fluoroscopic examinations of the upper airways with and without nasal CPAP. The epiglottis was large and lax, and upon positive pressure inspiration, it was literally blown down so it occluded the hypopharyngeal airway.

Bleomycin, methotrexate and vincristine before irradiation of stage III and IV laryngeal and pharyngeal squamous cell carcinoma. A study initiated by the Danish Society of Head and Neck Cancer.

Primary treatment with bleomycin, methotrexate and vincristine for two weeks followed by curatively intended 60Co irradiation was administered to 153 patients consecutively referred to the three main treatment centres in Denmark over more than a two-year period. Seventy-one laryngeal and 82 pharyngeal squamous cell carcinomas were evaluated. According to the TNM classification (UICC) 76 patients had stage III and 77 patients stage IV disease. The immediate response (complete + partial) to chemotherapy was 20 per cent. Judging from frequency of local recurrence, metastases as well as survival the treatment results were not obviously improved. A high frequency of complications was observed after this combination of chemotherapy and irradiation, and it was often impossible to fulfil the irradiation to the planned dose in appropriate time.

Medroxyprogesterone acetate and prednisone in advanced breast cancer. A randomized trial.

In a randomised trial patients with progressive metastatic breast cancer were allocated to one of three different treatments. A: Prednisone 10 mg X 3 daily. B: Medroxyprogesterone acetate (MPA) orally 500 mg daily. C: MPA i.m. 1000 mg daily for 3 weeks followed by 500 mg i.m. weekly. The study included 150 patients and was well-balanced with respect to different prognostic parameters. Most patients (83%) were postmenopausal, and 95% had previously received chemo- or hormonal therapy. In the MPA treated patients, analysis of serum MPA levels was performed once a month. The response rates were 4.6, 7.9 and 12.5% in treatments A, B and C, respectively. This difference was not statistically significant (P greater than 0.05). Furthermore, the follow-up of serum MPA levels revealed no significant difference between responders and non-responders. Analysis of time to progression did not indicate any advantage of MPA over prednisone, irrespective of MPA schedule. In accordance with these data, there was no difference as regards survival in the three groups. In conclusion, the study indicated that MPA is not superior to prednisone in this group of heavily pretreated patients with advanced breast cancer.

Standardized methyl green-pyronin Y procedures using pure dyes.

Fully standardized Methyl Green-Pyronin methods are presented. Pure Pyronin Y and purified Methyl Green or Ethyl Green are used either simultaneously in one dye bath or are used as a sequence of Pyronin Y and Ethyl or Methyl Green. Both methods, as shown by enzymatic pretreatment, give a reliable and reproducible staining in DNA with Ethyl or Methyl Green and of RNA with Pyronin Y on Carnoy fixed material. On formaldehyde fixed material it was found advantageous to use the sequential method as chromatin was hereby stained green instead of blue as seen with the simultaneous method.

Nucleic acid staining with the methyl green-pyronin method. A comparison of the use of pure dyes and commercially available dyes.

We compared the staining obtained using commercially available pyronin Y samples with that obtained using pure pyronin Y in a standardized methyl green-pyronin procedure. In addition, the importance of the dye content of the anhydrous dye was investigated by varying the dye content by the addition of pure pyronin Y to one of the commercially available pyronin Y samples. We found that, for routine histological work, commercially available pyronin Y samples may produce acceptable results provided the sample can be shown by spectrophotometry to contain at least 43% pyronin Y.