Analyses of protein extracts of human breast cancers: changes in glycoprotein content linked to the malignant phenotype.

The protein and glycoprotein composition of Triton X-100 extracts of breast biopsies from 17 women with benign breast disease and from 11 women with invasive breast carcinoma were investigated using electrophoresis in SDS-containing gradient polyacrylamide gels, followed by Coomassie Blue (CB) staining and the binding of radio-iodinated wheat germ agglutinin (WGA). Patterns were analysed after the CB-step for differences in protein composition, and after the WGA-step for differences in glycoprotein composition. Tissue extracts from patients with benign breast disease have less CB stained bands than similar extracts from the cancer patients. A particular consistent change was the appearance of an extra band at 58 Kdaltons in the cancer extracts. In contrast to the CB results, WGA detected less major bands, in the 40-60 Kd region, in the cancer extracts than at similar locations in benign extracts. Analysis of blood sera using the above techniques suggested that certain serum proteins could account for some of the WGA changes, but not the changes after CB staining. However, residual contamination of the specimens by blood proteins seemed unlikely because of the washing procedure used, unless these components were very strongly associated with the tissue. Differential synthesis of serum proteins by benign and malignant breast tissue may also explain some of our findings. Examination of the histopathology adjacent to the extracted tissue suggested that the degree of reduction in WGA-binding may be related to the extent of local invasiveness. Other animal and human studies suggest that reduced glycosylation of tumour-associated proteins may be linked to increased malignancy. The current findings may reflect a general pattern of change in tumour glycoprotein composition linked to malignant expression.

Polyoma virus transformation and two-dimensional electrophoresis patterns of baby hamster kidney cell native proteins.

The protein extracts of baby hamster kidney cells (BHK 21/C13) before and after malignant transformation by polyoma virus were resolved by isoelectric focusing in the first dimension and electrophoresis on concave 4-24% (w/v) polyacrylamide gradient gels in the second dimension. The resulting two-dimensional patterns were visualised by ultrasensitive silver staining. Major differences were observed in the protein distribution patterns of the two cell lines. Brief trypsinisation of the cells, prior to extraction of cell protein, had little effect upon the protein patterns of BHK cells. Similar treatment of the transformed cells markedly reduced the staining intensity of a string of spots which were present in far greater abundance than in the original BHK cells. The findings suggest that these proteins are located on the surface of the transformed cells and probably under the surface prior to transformation.

Serial use of aprotinin and incidence of allergic reactions.

Allergic reactions to aprotinin, a polypeptide, have been reported on several occasions. A survey of 136 courses of aprotinin, given to patients with gastric cancer as a protease inhibitor, has shown an incidence of less than 1% of acute anaphylaxis and a similar incidence of minor allergic responses. Two cases of acute allergic responses are described. Fifteen patients received repeated courses of aprotinin at 6-weekly intervals and no allergic phenomena were detected. Provided patients are challenged intra-dermally or with eye drops before each course, it is considered that aprotinin is safe to use in repeated fashion.

Cyclic GMP in urine to monitor the response to ovarian cancer to therapy.

Guanosine 3':5'-phosphate (cycle GMP) in urine has been used to monitor the response of patients with ovarian cancer to treatment. Changes in the cyclic GMP level appear to correlate well with clinical status in that the disappearance of clinically detectable tumour is associated with a drop in the level whereas a tumour recurrence is associated with an elevation. Serially measured cyclic GMP is valuable for detecting a recurrence of tumour growth in patients in clinical remission and can predate any clinical signs by as much as 10 months. In patients who show no response to treatment, cyclic GMP levels in urine are elevated in the majority of specimens collected.

Urine cyclic nucleotide concentrations in cancer and other conditions; cyclic GMP: a potential marker for cancer treatment.

Cyclic guanosine 3',5' monophosphate (cyclic GMP) and cyclic adenosine 3',5' monophosphate (cyclic AMP) have been determined in random urine specimens from 95 healthy individuals, 60 patients with non-cancerous conditions, 52 patients with benign tumours, and 74 patients with malignant tumours. Concentrations of cyclic GMP have also been determined in a number of other groups, including some undergoing cancer treatment. Ninety-three per cent of cancer patients had raised urinary cyclic GMP concentrations compared to the reference range for healthy subjects. For the non-cancerous and benign groups, 33% and 42% respectively had raised concentrations. The urine cyclic AMP concentrations were similar in all groups. Urine cyclic GMP appeared to rise early in the onset of malignant growth. Successful cancer treatment was accompanied by a dramatic fall in the urine cyclic GMP concentrations, whereas if the treatment was unsuccessful the level did not change. It is concluded that urine cyclic GMP may have important applications in the monitoring of cancer treatment.

Levels of adenosine 3',5' cyclic monophosphate and guanosine 3',5' cyclic monophosphate in single urine specimens collected from a large population of healthy subjects.

The levels of adenosine 3',5' cyclic monophosphate and guanosine 3',5' cyclic monophosphate have been measured in random urine specimens provided by 95 healthy volunteers with a view to assessing the importance of the effects of certain physiological factors on the cyclic nucleotide levels in this type of specimen, and to establish suitable reference ranges for future patient monitoring studies. Of the factors investigated, only the stage of the menstrual cycle was found to have a significant effect; even this variation was still within the overall normal reference range. Measurements of urine cyclic nucleotides were also made in relation to the method of specimen collection, handling, and storage. From these studies it was concluded that if the specimen is placed in a refrigerator within a few hours of collection and then transferred to a deep-freeze within 48 hours, these factors do not present any major problems for the subsequent assay of the cyclic nucleotides.

The role of cell surface proteins in the induction of histogenetic differentiative responses by some human breast tumours and hamster tumors implanted into chick embryos.

Cells of human breast tumours and fibrocystic hyperplasia grown in culture, and three hamster tumours were implanted between the cell layers of 18-hour-old chick blastoderm. Their ability to induce histogenetic responses in the ectodermal and endodermal embryonic tissues was investigated. The surface proteins of these tumour cells were labelled by lactoperoxidase-catalysed radioiodination. It is shown that the ability to induce the histogenetic effects may be related to the expression of 265K (K = 10(3) daltons) and 233K proteins on the surface of human tumour cells and of 115K proteins on the hamster tumour cells. The antiproteinase, aprotinin, inhibits the induction of the histogenetic responses by and apparently also prevents the deletion of 115K proteins from the hamster tumour cells. It is therefore suggested that cell surface proteins are involved in the complex processes of interaction between embryonic and tumour cells and in the recognition by the embryonic cells of the tumour cells implanted into their midst.

The effect of carcinoma of the cervix on the L-lactate-nicotinamide-adenine dinucleotide oxidoreductase activity in vaginal aspirates.

Lactate dehydrogenase (L-lactate-NAD+ oxidoreductase; EC 1.1.1.27) has been assayed in saline extracts of vaginal aspirates obtained from patients with carcinoma in situ and from patients with clinically detectable invasive carcinoma of the cervix. Taking premenopausal and postmenopausal patients separately, significant differences were found between the aspirate lactate dehydrogenase activities obtained from patients with carcinoma in situ and the activities obtained from patients with invasive carcinoma; generally higher activities were obtained from the latter. With the exception of postmenopausal patients with carcinoma in situ, aspirate enzyme activities obtained from both groups of patients were significantly different from those obtained from normal women. Significantly higher lactate dehydrogenase activity was found in aspirates from patients for whom histology showed the presence of an adenocarcinoma or certain highly malignant cell types. Treatment by radium insertion usually resulted in a fall total lactate dehydrogenase activity. Significant differences between groups were found for the proportions of single lactate dehydrogenase isoenzymes in a few instances but no significant difference in overall isoenzyme distribution was found.

Surface properties of cells isolated from non-metastasizing and metastasizing hamster lymphosarcomas.

Present evidence suggests that the cell surface has an important role in metastasis. To examine this idea further, the surface properties of single cells isolated from the primary growths of a liver-metastasizing (ML) and a non-metastasizing (NML) lymphosarcoma were compared for adhesion to cell monolayers, cytopherometry, isoelectric focusing, adhesion to immobilized lectins and surface labelling with lactoperoxidase-catalysed radioiodination. It was found that the ML cells had increased adhesion to 3 out of 4 of the monolayers studies; a lower overall surface charge but greater peripheral concentrations of charge; and increased surface expression of the fucose moiety. No consistent difference between the two cell types was detected in the electrophoretic pattern of the labelled surface proteins. These findings are discussed in the light of present knowledge of the cell surface, and it is concluded that the significance of any of the observed changes in relation to metastasis has yet to be established.

A simplified technique of high resolution two-dimensional electrophoresis: serum immunoglobulins.

A relatively simple, inexpensive technique is described for high resolution two-dimensional electrophoresis of human serum proteins. The technique clearly resolves the heavy chains of IgG, IgA and IgM, the positions of which have now been established. The technique is highly reproducible for normal serum. Distinct changes were observed upon examination of the sera of patients with IgG OF IgA myelomatosis or Waldenström's macroglobulinaemia. The technique should prove valuable in both the study of immunoglobulin disorders and studies into the fine structure of highly purified immunoglobulins.

The in vitro effect of aprotinin upon spleen cells from normal and tumour-bearing mice exposed to PPD and tumour cells.

Aprotinin (Trasylol) is shown to enhance the response of spleen cells from normal and tumour bearing mice to PPD nd tumour cells. This enhancement is greater in the tumour-bearing mice.

The effect of the menopause on the L-lactate-nicotinamide-adenine dinucleotide oxidoreductase activity in vaginal aspirates.

Lactate dehydrogenase (L-lactate-NAD+ oxidoreductase; EC 1.1.1.27) activity has been assayed in saline extracts of vaginal aspirates obtained from patients attending a Well Woman Clinic and for whom exfoliative cytology was shown to be unequivocally normal. The majority (76 per cent) of premenopausal patients gave aspirate extracts containing less than 25 m units of enzyme while 71 per cent of postmenopausal patients gave aspirate extracts containing more than 50 m units of enzyme. This difference in distribution of activities was highly significant (P = less than 0.001). Analysis of the isoenzyme distribution showed that aspirates from postmenopausal patients contained a lower proportion of isoenzyme LDH-5 (P less than 0.01) and the M subunit (P less than 0.03) and a higher proportion of isoenzymes LDH-3 (P less than 0.001) and LDH-4 (P less than 0.01). There was no significant difference between the social class distributions of the pre- and postmenopausal patients. Lactate dehydrogenase activity was not related to social class or patients' multiparity. No difference in the distribution of enzyme activities was found between those premenopausal patients in the proliferative phase of the menstrual cycle, those in the secretory phase and those receiving hormone therapy.

Immunological identity of urinary alkaline phosphatase in health and disease.

Urinary alkaline phosphatase has been measured and an investigation has been undertaken involving starch gel electrophoresis after treatment of the enzyme with antisera raised against various human tissues. Urine was obtained from patients with bone and liver disease as well as pregnant women and normal persons. The enzyme level in urine in disease and pregnancy was raised above the normal but considerable overlap occurred. After electrophoresis the most usual finding was a single zone of activity although occasionally very minor zones were also present. Antiserum to kidney had the greatest affinity for the urinary enzyme. Decreasing affinity was found with intestinal and placental antiserum. No affinity was found with liver antiserum.

Surface protein distributions in cells isolated from solid tumours and their metastases.

Methods have been developed which isolate single viable cells from the primary growths of two tumour systems (a lymphosarcoma and a carcinoma) and their secondary deposits. Subsequent comparisons of the surface-membrane structure of pairs of these primary and secondary cells, using lactoperoxidase-catalysed radioiodination coupled with polyacrylamide-gel electrophoresis, suggest that their overall structures are qualitatively very similar. This latter picture is still maintained when the isolated cells are treated with trypsin or incubated in complete medium before radioiodination. Analysis of the incorporated label into defined sections of the electrophoretic patterns revealed small quantitative differences between primary and secondary cells. In particular, slightly reduced incorporation into certain surface components of secondary cell preparations was seen. However, these did not occur for all the animals investigated, and also they did not consistently occur if the isolated cells were incubated in complete medium. The most similar overall change observed for the two tumour systems was a slight reduction in the secondary cells of a 20K mol. wt surface component.

Cyclic adenosine 3', 5' monophosphate: a possible indicator of premalignant changes in the large bowel.

Cyclic adenosine 3', 5' monophosphate (cyclic-AMP) has been estimated in mucosal biopsy samples removed from the descending colon and rectum at endoscopy to investigate the possibility of using this substance for monitoring pre-malignant changes in the large bowel. Four groups of patients have been studied: those with normal large bowel and rectal mucosa; those with non-malignant inflammatory bowel disease; those with an adenomatous polyp in the descending colon or sigmoid colon; and those with a rectal adenocarcinoma. No difference was found in the cyclic-AMP content of 'normal' rectal mucosa, 'normal' colonic mucosa, 'diseased' colonic mucosa, carcinomas, and uninvolved mucosa adjacent to the polyps. Less cyclic-AMP was found in the polyps than in adjacent uninvolved mucosa. Conversely, more cyclic-AMP was found in the carcinomas than in adjacent uninvolved mucosa. It is concluded that although cyclic-AMP may be a very useful parameter for delineating the extent of the disease in individual patients, it is not a suitable biochemical marker for the screening of neoplastic changes in the large bowel in the population as a whole.

Aprotinin induces surface changes in malignant cells in culture: a possible mode of antitumour action.

Treatment of TRES cells with aprotinin (Trasylol) produced marked changes in their surface properties. There was a concentration-dependent increase in the surface charge density of the cells. The agglutinability of the cells was inhibited by the antiproteinase. The cells became less adhesive to concanavalin A-linked plates. The adhesion of treated TRES cells appeared to be mediated by lectin receptors with lower specificity for concanavalin A. These observations and an analysis of the kinetics of adhesion have suggested the possibility that a new class of concanavalin A receptors appears on the surface of aprotinin-treated TRES cells, which might increase immunogenicity of the cells.

Increased nuclear size in BHK21 cells after treatment with non-toxic levels of calf thymus histones.

The effect of treatment with either whole calf thymus histones, or individually isolated histones, or polyarginine, or polylysine, on the nuclear size of BHK21 cells has been investigated. Incubation of the cells with mixed histones (12.5--44 microgram/ml) for 1 h considerably increased nuclear size. Increasing the histone concentration and/or the incubation time resulted in a decrease in the effect and could result in no change in nuclear size. Treatment of the cells with polyarginine or polylysine did not affect nuclear size. Experiments with individually isolated histones showed that the nuclear size effect was almost exclusively due to the histone H4. It is argued that the changes observed most likely resulted from interaction of H4 with the nucleus, and could reflect the properties of this particular histone molecule.