High-resolution proton nuclear magnetic resonance analysis of metastatic cancer cells.

High-resolution proton nuclear magnetic resonance (NMR) studies of intact cancer cells revealed differences between cells with the capacity to metastasize and those that produce locally invasive tumors. The NMR resonances that characterize the metastatic cells were associated with an increased ratio of cholesterol to phospholipid and an increased amount of plasma membrane-bound cholesterol ester. High-resolution NMR spectroscopy could therefore be used to assess the metastatic potential of primary tumors.

Critical modulation by thymidine and hypoxanthine of sequential methotrexate-5-fluorouracil synergism in murine L1210 cells.

Treatment of murine L1210 cells with methotrexate (MTX) followed by 5-fluorouracil (FUra) produced synergistic cytotoxicity, but only in media containing serum with low concentrations of hypoxanthine, such as horse serum and dialyzed fetal calf serum. Addition of hypoxanthine (1 to 10 microM) during drug exposure reduced the synergism of sequential MTX (1 to 100 microM)-FUra (30 to 300 microM) treatment. The reduction of synergy by hypoxanthine varied with the MTX concentration, higher hypoxanthine concentrations being required to prevent synergy at higher MTX concentrations. The cytotoxicity produced by sequential MTX (10 microM)-FUra (30 to 300 microM) treatment was also reduced if thymidine was added to the regrowth media following drug exposure. The rescue by thymidine was concentration dependent, but as little as 0.5 microM thymidine was sufficient to substantially reduce the synergistic cytotoxicity. These results indicate that both hypoxanthine and thymidine are critical determinants of sequential MTX-FUra synergy and call into question the relevance of experiments in low-thymidine- and low-hypoxanthine-containing media to the clinical situation, where plasma hypoxanthine and thymidine concentrations are often greater than 1 and 0.5 microM, respectively.

Diagnosis of follicular thyroid lesions by proton magnetic resonance on fine needle biopsy.

Most thyroidectomies are currently performed for diagnostic purposes. It has been established that proton magnetic resonance spectroscopy (MRS) on excised thyroid tissue can distinguish normal thyroid from invasive carcinomas (P < 0.0001). The purpose of this study was to assess whether the same discrimination could be obtained preoperatively from fine needle biopsy (FNB). This has clinical importance because cytological examination of fine needle aspirates cannot distinguish between benign and malignant follicular thyroid lesions. Here we demonstrate a sensitivity of 95% for proton MRS to correctly identify clinically or histologically proven carcinoma. MRS measurements were made on FNB specimens (containing as few as 10(6) cells) from solitary thyroid nodules. MR assessment of FNB was inconsistent with that of the corresponding tissue in only 6.5% of cases. The discrimination between cancer and normal tissue was based on altered cellular chemistry measured as a one-dimensional spectral ratio of resonances from the amino acid lysine and lipid. Benign follicular lesions were separated into two groups: 67% with a spectral ratio similar to malignant thyroid tumors, and 33% with a spectral ratio comparable to that in normal thyroid tissue. Thus, in contrast with histopathology, MRS offers a method for assessment of FNB of follicular lesions with the potential to identify a biologically benign group, which could avoid thyroid surgery for purely diagnostic purposes.

A proton magnetic resonance spectroscopy study of aging and transformed human fibroblasts.

Proton magnetic resonance spectroscopy (1H MRS) has been used to monitor changes occurring during aging and transformation in human lung fibroblasts. Aging was studied in MRC-5 cells from nonsenescent (early passage) to presenescent (late passage) and senescence. Nonsenescent cells infected with SV40 virus (pretransformed) were monitored through crisis and subsequent immortalization. Aging changes were observed with one- and two-dimensional MR spectra. Cholesterol and lipid resonances were significantly increased from nonsenescent cultures to senescence. These changes could be caused by chemical or structural changes in the plasma membrane or in intracellular lipid pools. In contrast, choline levels rose from nonsenescent to presenescent cells but at senescence dropped to that of nonsenescent cells. Increased choline levels are often associated with increased cellular proliferation. After SV40 infection of MRC-5 cells there was an increase of cholesterol and lipid levels that peaked at crisis. Newly immortalized cells exhibited a drop in cholesterol and lipid to nonsenescent cell levels, but these rose again in established immortalized cells. In contrast to presensescent cultures, the levels of choline gradually increased from pretransformed to crisis phase but still continued to rise after immortalization. Thus, 1H MRS illustrates similarities in lipid behavior at senescence and crisis, whereas the choline levels are different.

Isodicentric X chromosomes involving the Xq13 breakpoint in myelodysplasia and acute nonlymphocytic leukemia.

Many nonrandom chromosome abnormalities, usually autosomal in nature, have been found to be associated with specific types of acute nonlymphocytic leukemia (ANLL) and myelodysplastic syndromes (MDS). Specific abnormalities involving the sex chromosomes are rare. We have recently identified a structural abnormality of the X chromosome, for example, idic(X)(q13) in three patients: two with MDS progressing to ANLL and one with ANLL de novo. All three patients were elderly females with a very aggressive form of ANLL. Six other patients with a similar abnormality have been discovered in the literature; all having either MDS or ANLL and a short survival. It is suggested that the abnormality identifies a subset of MDS and ANLL occurring in elderly females.

Human cancers detected by proton MRS and chemical shift imaging ex vivo.

Proton magnetic resonance spectroscopy (1H MRS) has the potential to become a diagnostic adjunct for the detection and grading of human neoplastic disease. This paper describes the use of proton MRS to document changes arising in the lipid chemistry of biopsies arising from the human uterine cervix, thyroid and colon and demonstrates the diagnostic power of ex vivo spectroscopy. Proton chemical shift imaging (CSI) is further used to determine the spatial location of lipid changes in ex vivo human biopsy specimens and provides insight into the chemistry of neoplastic transformation.

Malignancy-related characteristics of wild type and drug-resistant Chinese hamster ovary cells.

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.

Characterization of human ovarian epithelial tumors (ex vivo) by proton magnetic resonance spectroscopy.

Proton magnetic resonance spectroscopy (1H MRS) offers an alternative investigational modality that will assist current pathologic techniques in the diagnosis of human ovarian epithelial tumors. Histologically normal human ovarian tissue (n = 12) was compared with ovarian benign fibromas (n = 3) and surface epithelial-stromal tumors (benign, n = 18; proliferating, n = 9; frankly malignant, n = 30) ex vivo by 1H MRS. The distinction between carcinomatous and benign or normal tissue (P<0.0001; Student's t-test) was made on one-dimensional (1-D) 1H MR spectra utilizing differences in resonance intensities of cellular lipid, creatine/phosphocreatine and lysine. The sensitivity and specificity of the test were 87% and 91%, respectively. Two-dimensional (2-D) MRS of carcinomatous biopsies showed multiple crosspeaks attributable to cell-surface fucosylation that correlated with tumor grade and loss of cellular differentiation. The multiple fucose crosspeaks were absent in spectra from normal ovary and benign tumors. The distinction between carcinomatous and normal or benign tissue based on MR-visible fucosylation gave a sensitivity and specificity of 88% and 97%, respectively. Proliferating tumors exhibited a range of cell-surface fucosylation patterns indicative of malignant potential.

NMR methods for characterizing the state of the surfaces of complex mammalian cells.

It is shown that narrow 1H NMR resonances may be observed in cancer cells, and that these belong to fatty acyl chains of membrane lipids. A variety of NMR techniques such as Gaussian-Lorentzian deconvolution, and T1 and T2 measurements, may be used to subdivide these resonances further. The results of these various methods require that in the membrane structures the observed lipids tumble isotropically and sufficiently rapidly to give motionally narrowed 1H NMR lines.

Proton magnetic resonance spectroscopy of lymphocytes: an historical perspective.

Proton magnetic resonance spectroscopy can be used to elucidate alterations to cellular chemistry associated with specific biological functions. It became apparent that this type of information was present in the magnetic resonance spectra from intact viable lymphocytes in the late 1970s. It was not until the 1980s, however, that one-dimensional multipulse sequences were used to filter the large signal contributions from water and fat, which had until then masked weaker signals from other molecules. When this technology was combined with two-dimensional spectroscopy, unambiguous assignment of the biologically relevant chemical species became possible. In vitro activated, stimulated, transformed, and malignant lymphocytes, as well as embryonic fibroblasts and malignant cells of epithelial origin, all gave rise to a strong triglyceride spectrum and resonances from a multitude of cellular metabolites. Two-dimensional spectroscopy and the analyses of highly purified membranes determined that the triglyceride signals originated, at least in part, from the plasma membrane. Based on physiochemical data, a new model for the structural arrangement of plasma membrane lipid in these cells was proposed. While differences exist between the proton magnetic resonance spectra of stimulated lymphocytes and malignant cells in vitro, they share a high-resolution lipid spectrum. In tissue, however, the presence of activated lymphocytes does not always produce the lipid spectrum, particularly in the vicinity of tumors.

Assessment of enzyme inhibition or stimulation in plasma membrane preparations.

The purity of isolated plasma membranes is routinely judged by the activity of enzymes present both in this membrane and other locations in the cell. However, since enzyme inhibition and/or stimulation often occurs following disruption of the cell, the question as to which enzyme(s) provides a reliable marker of membrane purity should be considered. We have devised a simple method with which to address this problem. Inhibition or stimulation of plasma membrane marker enzymes can be rapidly assessed in cell homogenates and subfractions by mixing both samples, with known enzyme activity, and observing any deviation from the expected combined activity. Should the activity remain constant that enzyme can be used to gauge the purity of the plasma membrane preparation. Of the four putative plasma membrane marker enzymes examined only one, gamma-glutamyltranspeptidase appeared to give a reliable purity measurement in the cell system studied.

Fucose in 1H COSY spectra of plasma membrane fragments shed from human malignant colorectal cells.

The methyl-methine cross peak of bound fucose has been assigned in the COSY spectrum of plasma membrane shed from human malignant colorectal cells. This cross peak (1.33-4.27 ppm), which is superimposed on the methyl-methine cross peak of threonine, was assigned following hydrolysis of the sample. Acid hydrolysis led to a 28 +/- 5% reduction in the intensity of the cross peak and the appearance of the alpha and beta forms of fucose. Chemical analysis confirmed the release of free fucose. Lactate anion, which was not perturbed by the hydrolysis, was used as an internal standard.

Esterified cholesterol and triglyceride are present in plasma membranes of Chinese hamster ovary cells.

The chemical composition of highly purified plasma membrane preparations from a series of malignant Chinese hamster ovary (CHO) cell lines were undertaken to ascertain if neutral lipid, including cholesteryl ester and triacylglycerol, were present. Triacylglycerols (33-41 nmol/mg total lipid) and cholesteryl ester (226-271 nmol/mg) were measured in the plasma membranes and differences in the chemical composition of these membranes recorded. The most significant difference was a gradual decrease in the level of free cholesterol from wild type (312 +/- 7 nmol/mg total plasma membrane lipid), Pod RII-6 (268 +/- 64 nmol/mg total plasma membrane lipid), Col R-22 (243 +/- 39 nmol/mg total plasma membrane lipid) to EOT (204 +/- 20 nmol/mg total plasma membrane lipid), with a concomitant increase in the degree of saturation of the cholesteryl ester fatty acids, particularly palmitic acid. No statistically significant differences were apparent in the chemical composition of the whole cells in this series. The one-dimensional (1D) 1H-NMR spectra of the four malignant cell lines showed a gradation in intensity of lipid resonances, in the order of wild type, Pod RII-6, Col R-22 and EOT, with EOT having the strongest lipid spectrum. Interestingly, the increase in acyl-chain signal intensities in the 1H-NMR spectra of this series of CHO cells and emergence of signals from cholesterol and/or cholesteryl ester, coincide with alterations in the amount of free cholesterol and the degree of saturation of the fatty-acyl chain of the esterified cholesterol in the plasma membranes. It is our hypothesis that, together, cholesteryl ester and triacylglycerol form domains in the plasma membrane and that when the cholesteryl ester has a largely saturated fatty acid content, the lipids are in isotropic liquid phase and hence visible by NMR.

Correlation of cellular differentiation in human colorectal carcinoma and adenoma cell lines with metabolite profiles determined by 1H magnetic resonance spectroscopy.

The aim was to determine whether proton magnetic resonance spectroscopy (MRS) could grade human colorectal cells of differing malignant potential. A cell model of tumour development and progression comprising 2 non-tumorigenic adenoma lines and 4 carcinoma lines of increasing tumorigenicity was chosen. A gradual reduction in cellular differentiation and an accumulation of genetic alterations from adenoma to carcinoma characterized the selected cell lines. One-dimensional and 2-dimensional MRS showed that reduced differentiation in the cell model correlated with an increase in the levels of lipid, metabolites, the glycosylation intermediate uridine diphospho-N-acetylglucosamine and cell-surface fucosylation. Mutations involving the K-ras, APC and DCC genes are present both in adenoma- and in carcinoma-derived lines in this model, but the first evidence of an abnormality in the p53 gene was concomitant with the cells' ability to grow as a tumour in athymic nude mice. This genetic change coincided with the detection, by MRS, of UDP-hexose (ribose moiety, 2D MRS cross peak between H2 at 4.38 ppm and HI at 5.99 ppm) and the appearance of an additional fucosyl resonance (cross peak between-CH3 at 1.41 and H5 at 4.30 ppm) in the least tumorigenic of the carcinoma cell lines. An increase in complexity of the fucosylation spectral pattern was observed with further cellular de-differentiation and increased tumorigenicity. Collectively these data support the existence of an adenoma-carcinoma sequence.

Evaluation of an in vitro invasion assay for use on solid tissue samples and cultured cells.

An invasion assay, developed for monitoring the in vitro penetration of reconstituted basement membrane, matrigel, was modified and successfully applied to solid tumours, normal tissues as well as a variety of normal and tumour cell lines. However, we found that some normal fibroblasts were capable of in vitro invasion whilst some malignant cell lines with invasive capacity in vivo did not penetrate the matrigel. Nevertheless, this method can distinguish invasive capacity within a tumour model, and as a consequence may be used to elucidate some of the biochemical mechanisms in the invasion process comparing cells grown both in vitro and in vivo. Since the method does not always correlate with invasion in vivo the results must be interpreted with caution.

Further evidence that the narrow 1H magnetic resonance signals from malignant cells do not arise from intracellular lipid droplets.

1H magnetic resonance (MR) spectroscopy of intact viable malignant cells yields high resolution spectra from lipid. In previous studies we have provided evidence that these signals are generated by neutral lipid located in the plasma membrane in unique domains. We show that intracellular lipid droplets do not contribute to the MR signal. Two malignant Chinese hamster ovary cell lines, EOT and its parental line WT were studied. The EOT cells have a more highly resolved lipid spectrum than the WT, a result which correlates with slightly increased levels of triglyceride in highly purified plasma membranes. The intracellular lipid droplets of both lines were quantified using both fluorescence and electron microscopy but no significant differences were observed. Together these results provide evidence that narrow 1H MR signals from malignant cells arise from neutral lipid in the plasma membrane, rather than from intracellular lipid droplets.

Magnetic resonance spectroscopy detects cancer in draining lymph nodes.

The spread of cancer cells to draining lymph nodes is an important prognostic factor for many cancers and influences postoperative therapy in patients. Histopathology is used routinely to assess if lymph nodes contain metastases. There are, however, time and resource constraints on the volume of lymph node tissue that can be examined by the pathologist in a routine laboratory (less than 2% of each node), thus major sampling errors are possible. Conventional histopathology also relies on identifying aggregates of malignant cells for a positive diagnosis. Proton (1H) magnetic resonance (MR) spectroscopy can detect chemical changes, specifically increased levels of lactate, choline, fucose and amino acids, in lymph nodes infiltrated by cancer. Increase in lactate indicates the presence of anaerobically respiring cells, whereas choline reports that the cells are replicating. Since MR spectroscopy can identify early infiltration by malignant cells, before cell clusters are visible under the light microscope, it detects micrometastases in lymph nodes missed histopathologically. Furthermore, MR spectroscopy eliminates sampling errors since the entire lymph node is examined.

Cell-surface fucosylation and magnetic resonance spectroscopy characterization of human malignant colorectal cells.

Proton (1H) magnetic resonance spectroscopy (MRS) has been used to distinguish lowly and highly tumorigenic human malignant colorectal cell lines based on differences in lipid, choline, and fucose resonances. The spectral patterns were comparable with those obtained for human colorectal biopsy specimens, indicating that cells grown in vitro are suitable for documenting colorectal tumor biology. For the first time, two-dimensional (2D) correlation spectroscopy (COSY) has been used to assess the fucosylation state on the surface of intact viable cells, and differences were recorded between the highly and lowly tumorigenic cell lines. Four methyl-methine cross-peaks were assigned to covalently linked fucose on the basis of increases in volume following the addition of free fucose. Both cell lines incorporated the same amount of exogenous free fucose as determined chemically, but the COSY spectra indicated that the fucose was distributed differently by each cell line. Of the four sites containing MR-visible bound fucose, one was common to both cell lines, two characteristic of the highly tumorigenic line, and the remaining site unique to the lowly tumorigenic cells. Material released from the highly tumorigenic cells in response to increased cell density was also fucosylated (whereas shed material from lowly tumorigenic cells was not), suggesting a biological role for shed fucosylated antigens in tumor aggression.