Marlen Dyne

Proteolipid identified by magnetic resonance spectroscopy in plasma of a patient with borderline ovarian tumour

Magnetic resonance spectroscopy (MRS) can identify abnormal lipoproteins in the plasma of patients with premalignant and malignant tumours. Proteolipid complexes, 8-11 nm and 25-28 nm in size, were isolated from the plasma of a patient with a borderline ovarian tumour. These complexes, which generated a characteristically long MRS T2 relaxation value (greater than 400 ms), were disrupted by ribonuclease. None of the conventional lipoproteins had a T2 value above 160 ms. Chemical analysis of the proteolipid complexes showed a 20% glycolipid component, and MRS identified a fucosylated molecule as the origin of the long T2 value. 9 months after resection of all tumour, a visible lipoprotein band, possibly lipoprotein (a), persisted in the plasma but neither the long T2 relaxation value nor the 8-11 nm or 25-28 nm particles were present. The long T2 relaxation value in the MRS profile, found in isolated proteolipid and unfractionated plasma and serum of other patients with carcinoma of the ovary and colon, provides a non-invasive method of assaying for cancer.

A proteolipid in cancer cells is the origin of their high‐resolution NMR spectrum

High‐resolution proton nuclear magnetic resonance studies show that the spectrum of a proteolipid complex, isolated from the serum of patients with malignant diseases, is directly comparable with that obtained from intact cancer cells and solid tumours. These NMR signals have previously been shown to reveal differences between cancer cells with various biological characteristics such as metastatic capacity and drug sensitivity. The proteolipid contains cholesterol, phospholipid, triglyceride, glycolipids, ether‐linked lipids, and an apoprotein of unusual electrophoretic mobility. We have yet to confirm the presence of the mRNA reported by others. NMR spectroscopy could be used as a rapid method of identifying the presence of this proteolipid complex in human serum and aiding the diagnosis of malignant disease.

Lipid domain in cancer cell plasma membrane shown by 1H NMR to be similar to a lipoprotein

Human blood lipoproteins have been characterised by 1H NMR methods and chemical analysis, and comparisons made with the properties of the triglyceride‐rich plasma membrane domain found in cancer cells. By means of selective and non‐selective T 1 experiments, the lipids in HDL and LDL are shown to be in diffusive exchange. In contrast, the lipids ofchylomicra and VLDL do not exhibit lipid diffusion, and therefore resemble the neutral lipids of cancer cell plasma membranes. 2D scalar correlated NMR (COSY) spectra of cancer cells or solid tumours are similar to those obtained from VLDL and LDL. The long T 2 relaxation value observed for neutral lipid methylenes in metastatic cancer cells (>00 ms) was not observed for any of the 4 lipoproteins studied. None of the lipoprotein classes gave a T 2 longer than 250 ms.

Tubular sodium handling and tubuloglomerular feedback in compensatory renal hypertrophy

Tubular sodium handling and tubuloglomerular feedback (TGF) activity were assessed in established compensatory renal hypertophy in Sprague Dawleys rats. Hyperfiltration at the level of the single nephron was confirmed 4–6 weeks following a reduction in renal mass. TGF activity, determined as the difference between late proximal and early distal measurements of single-nephron glomerular filtration rate (SNGFR), was significantly increased in compensatory renal hypertrophy, being 7.8±1.0 vs 23.3±1.9 vs 25.5±2.6 nl/min (P for analysis of variance <0.05) following sham operation, unilateral nephrectomy, and 1 1/3 nephrectomy, respectively. Enhanced net tubular Na transport was also observed, with total Na reabsorption up to the late proximal site being 1.8±0.2 vs 2.7±0.1 vs 3.1±0.3 nmol/min (P<0.05), and to the early distal site being 3.4±0.5 vs 5.8±0.6 vs 7.9±0.8 nmol/min (P<0.05) in the three animal groups respectively. Comparison of proximal tubular length demonstrated a 71.9±8.1% increase in uninephrectomised vs sham-operated animals. This increase was proportionately greater than the increase in proximal Na reabsorption (50.0±4.0%) observed in the corresponding animal groups. Concurrent electron microprobe experiments in uninephrectomised and sham-operated animals demonstrated that the proximal tubular intracellular Na concentration was significantly lower following uninephrectomy (16.8±0.6 vs 18.9±0.5 mmol/kg wet weight, P<0.01), in association with evidence of reduced basolateral Na/K-ATPase activity. In summary, these data indicate that total Na transport in individual nephrons is increased in the proximal tubule and in the loop of Henle in compensatory renal hypertrophy, although the net amount of Na reabsorbed per unit proximal tubular length is actually reduced. The cell composition data suggest that the site of inhibition of transcellular transport is at the apical cell membrane. The elevated SNGFR is under the regulatory influence of an appropriately activated TGF system, which serves to limit the hyperfiltration.

Cell surface involvement in cancer metastasis: an NMR study

NMR spectroscopy is one of the few techniques which has the sensitivity to detect subtle changes to the surface chemistry of cells. It has previously been demonstrated that high resolution 1H NMR methods can distinguish tumour cells with the capacity to metastasise and this information appears to arise from a type of proteolipid in or attached to the plasma membrane. Here we report that the 1H NMR signal, which we have used to identify metastatic cells in rat tumours, is significantly reduced in intensity after cultured cells are treated with trypsin/EDTA. The long T 2 relaxation value (⪢ 350 ms) observed in metastatic cells is absent after enzyme treatment. 2D scalar correlated NMR (COSY) spectra of these treated cells show that a cross peak normally associated with malignancy and metastatic disease is markedly reduced. These findings indicate that the plasma membrane lipid particle which generates the high resolution spectrum is directly affected by trypsin/EDTA. Alterations to the cell surface properties were also demonstrated in vivo since reduced numbers of metastases were observed in animals injected with enzyme‐treated cells. The correlation between the absence of a long T 2 relaxation value and the diminished numbers of metastases in animals suggests that the plasma membrane particle is involved in the metastatic process.

Proximal tubular cell sodium concentration in early diabetic nephropathy assessed by electron microprobe analysis

Electron microprobe X-ray analysis techniques were employed in order to assess the changes that occur in proximal tubular cell sodium concentration during the hyperfiltration phase of early diabetes mellitus induced by streptozotocin in Sprague Dawley rats. Intracellular rubidium accumulation following intravenous infusion of rubidium chloride was used as a marker of basolateral Na/K-ATPase activity. The diabetic animals studied had a significantly higher glomerular filtration rate compared with controls [1.44±0.07 vs. 1.00±0.07 ml min−1 (100 g body weight)−1; mean±SEM, P<0.001]. Intracellular Na concentration was significantly higher in diabetic animals (19.5±0.6 vs. 17.8±0.4 mmol/kg wet weight; P<0.01). Concurrent measurement of Rb demonstrated significantly higher intracellular accumulation in the proximal tubules of diabetic animals compared with control (7.9±0.5 vs. 5.5±0.5 mmol/kg wet weight; P<0.001). These results indicate that proximal tubular Na/K-ATPase activity is enhanced in the hyperfiltration phase of diabetes mellitus. Since, however, intracellular Na concentration is increased under these conditions, it may be inferred that apical Na entry into proximal tubular cells is stimulated beyond the rate of basal exit during the initial development of hyperfiltration. The reasons for these alterations in cellular Na transport are unclear but similar changes have been implicated in the pathogenesis of cell growth.

Vinblastine sensitivity of leukaemic lymphoblasts modulated by serum lipid

The high‐resolution proton magnetic resonance spectrum of leukaemic lymphoblasts is characteristic of neutral lipid in an isotropic environment. When such lymphoblasts are selected for resistance to the anticancer drug vinblastine, the intensity of this spectrum increases with increasing drug resistance. A reversal of this trend can be achieved by growing cells in delipidated serum, whereby lipid spectrum and drug resistance are diminished. However, both can be restored by subsequent regrowth in normal medium. Thus, although detectable genetic changes accompany the development of vinblastine resistance, the expression of these changes can be modulated by environmental lipid.

MALIGNANCY-RELATED CHARACTERISTICS OF WILD TYPE AND DRUG-RESISTANT CHINESE HAMSTER OVARY CELLS

&NA; Chinese hamster ovary (CHO) cell lines are a very popular cell model for a wide range of studies but are often misused experimentally as a substitute for normal cells. Although CHO was originally derived from normal tissue, the cell lines studied here, including the parental wild type, have many characteristics which indicate that they have undergone malignant transformation. Biological properties associated with malignancy were investigated in this study on wild type CHO cells and 4 drug resistant sublines, EOT, Col R‐22, Pod R11‐6, and Vin R‐1. We report evidence of tumorigenicity in experimental animals, invasive capacity, in vivo and in vitro, protease release by 2 of the cell lines, features related to drug resistance in the mutant sublines, and numerical and structural chromosomal abnormalities.

Electron probe X‐ray microanalysis of intracellular element concentrations in cryosections in the presence of changes in cell volume

The interpretation of element concentration data for X‐ray microanalyses of biological tissues, which are subjected to some experimental treatment, can be complicated by changes in cell volume and total cell dry matter induced by the treatment. We have examined the manner in which such changes would affect the values measured in frozen‐dried cryosections of soft tissues, and how they may be taken into account in the interpretation of the results. The element content (mass per unit dry weight) measured by the peak‐to‐continuum or Hall method is independent of changes in cell volume, but is sensitive to a change in the local dry mass. Conversely, intracellular concentrations in terms of mass per unit volume, as determined by the peripheral or internal standard technique, are dependent on volume changes but independent of dry mass. The estimated dry weight fraction is affected by changes in both volume and dry mass. The results obtained from both quantification methods can therefore provide information on the combination of changes in cellular element levels, volume and total dry mass that may occur following the experimental treatment.

Proximal tubular cell electrolytes during volume expansion in the rat.

1. Proximal tubular intracellular elements were measured by electron microprobe X‐ray analysis (a) in rats volume‐expanded with albumin‐saline in which peritubular oncotic pressure remained normal and (b) in rats in which the renal artery was snared before volume expansion (the early snare model). Glomerular filtration rate and urine Na+ excretion were measured in addition to intracellular Rb+ following a 30 s infusion of RbCl as a marker for K+ transport. 2. In albumin‐saline volume‐expanded rats, intracellular levels of Na+ ([Na+]i) at 21.5 +/‐ 0.6 mmol (kg wet wt)‐1, Cl‐ ([Cl‐]i) at 18.0 +/‐ 0.4 mmol (kg wet wt)‐1 and Rb+ ([Rb+]i) at 9.4 +/‐ 0.4 mmol (kg wet wt)‐1 were significantly higher (P < 0.0001) than the levels in non‐expanded rats ([Na+]i, [Cl‐]i and [Rb+]i at 17.7 +/‐ 0.4, 14.6 +/‐ 0.3 and 4.7 +/‐ 0.4 mmol (kg wet wt)‐1, respectively; means +/‐ S.E.M.). The data are consistent with Na+ pump inhibition in the proximal tubule, although this cannot be directly derived from intracellular element measurements. 3. In an early snare model of volume expansion, [Na+]i, intracellular K+ ([K+]i) and [Rb+]i remained unchanged (16.1 +/‐ 0.4, 131.0 +/‐ 2.0 and 5.2 +/‐ 0.3 mmol (kg wet wt)‐1, respectively) compared to non‐expanded snared kidneys (15.9 +/‐ 0.6, 131.3 +/‐ 1.8 and 4.8 +/‐ 0.3 mmol (kg wet wt)‐1, respectively). [Cl‐]i at 18.3 +/‐ 0.5 mmol (kg wet wt)‐1 increased (P < 0.0008) compared to controls at 15.8 +/‐ 0.5 mmol (kg wet wt)‐1. Thus, in these rats, evidence for an inhibition of the Na+ pump was no longer observed. This points to a major intrinsic mechanism within the kidney for mediating natriuresis, since circulating factors were identical to those in the unsnared kidney, where significant natriuresis occurred.