Ann. din. Biochem. 13 (1976) 458-466
Wellcome Award Lecture
A. L.
LATNER
University Department of Clinical Biochemistry, Royal Victoria Infirmary, Newcastle upon Tyne, NEl 4LP
It is indeed a great honour to receive the Wellcome
Latner and Hodson, 1961; Hodson et al., 1962; award, and I do this in all humbleness knowing quite Latner, 1965; Latner et al., 1970a, 1970b; Hodson well that much of my work has depended on colla- and Latner, 1975), as well as with the arninotransboration with a number of younger colleagues. In ferases (Boyde and Latner, 1962). Human LD-l has particular I wish to mention Dr. A. W. Skillen, been isolated in pure form as microcrystals (Emes Dr. G. A. Turner, Dr. Gordon Dale, Dr. A. V. Emes, et al., 1974) and I am now able to announce the and Mr. A. W. Hodson, although there have been isolation, also in pure form, of human liver alkaline phosphatase (Hodson and Latner, unpublished quite a number of other collaborators. The Wellcome award is given particularly for work observations). This was done by us more than a year related to the quality of laboratory practice. This, of ago. course, involves the development of new techniques, I first developed the concept that isoelectric and I should like to emphasise that technical focusing could be carried out in small gel rods at a development can be of two types. In the first, the time when I was watching a salesman's demonstrapurely analytical, there is the development of a tion of the large column in which focusing is done in technique of estimation, manual or automatic, which liquid. It occurred to me that a gel rod technique can be the means of estimating a substance, analysis would be not only more rapid but considerably more for which has not been previously available, or economical. This idea led to the co-operative work alternatively can improve such things as specificity, with Dr. Dale which culminated in the first publicaprecision, accuracy, cost, speed of determination, tion in the world literature of the technique of isoand the like. This type of development can in itself electric focusing in acrylamide gel (Dale and Latner, be a source of great satisfaction and a great help 1968). for the routine diagnostic laboratory. In this My studies with isoenzymes, isoelectric focusing, respect, I have had the good fortune to be involved and saturation assay using radioisotopes, I am happy in a number of instances. These have included the to say, have opened up a number of interesting estimation in body fluids of the therapeutic sub- fields. For the purpose of this lecture, however, I stance Miracil (Coxon et al., 1947), of ergothioneine have decided to discuss their relationship only to in human blood (Latner, 1948), a simple emergency cancer research carried out in my department. method for the estimation of blood glucose (Latner and Pendlenton, 1949) and the estimation of magneTECHNICAL METHODS sium by atomic absorption spectrophotometry (Horn and Latner, 1963), and, like most of us, an lsoenzymes even larger number of unpublished contributions. The second type of technical development is the Separation of isoenzymes is effected by vertical deliberate elaboration of methods intended solely starch-gel electrophoresis (Smithies, 1959) in starch for the purpose of investigation of a variety of blocks, 30 ern long, 0.6 ern thick, and of varying disease processes, not only in relation to diagnosis width, depending upon the number of samples to be but also to mechanisms of development of the examined. The samples are either sera or aqueous disease process itself. I personally have obtained extracts of tumours or normal tissues. The buffer even greater pleasure from this situation. Isoenzyme employed is either 0.05 M Tris-HCI, pH 8.6 (Latner studies, isoelectric focusing, and saturation assay and Skillen, 1961), or 0.025 M Tris-borate-EDTA at helve proved particularly rewarding in this respect. the same pH. A voltage gradient of 6 V/cm is Lactate dehydrogenase has been the isoenzyme applied for 2 hours. On completion of electrosystem mostly studied, by means of starch gel phoresis the gel is sliced into five slices, each of electrophoresis, using a modified Smithies's method which can be stained in a different way. To demon(1959) and a specific staining technique for enzyme strate lactate dehydrogenase (LD) isoenzyme bands activity (Latner and Skillen, 1962; Latner and a tetrazolium salt (MTT) is used, together with Turner, 1967; Latner and Skillen, 1968). Success phenazine methosulphate, NAD, and sodium has also been achieved with the isoenzymes of lactate in 0.3 M Tris-HCl buffer, pH 7.4. After human alkaline phosphatase (Hodson et al., 1961; incubation at 37°C the isoenzyme zones appear as 458
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Wellcome award lecture
purple bands due to formazan formation. Other dehydrogenase isoenzymes can be demonstrated in a similar manner by using the appropriate substrate (Latner and Skillen, 1968).
lsoelectric focusing followed by electrophoresis Both these procedures are carried out inacrylamide gels. Isoclectric focusing has been performed in an acrylamide gel rod, using Ampholine to produce the pH gradient. When focusing is complete, the gel rod is placed on top of a buffered gel block, into which the focused proteins are made to travel at pH 8.6 by means of electrophoresis. They can then be stained by an appropriate stain (Dale and Latner, 1969). We now prefer to use Coomassie brilliant blue G. The electrophoresis block can be a gradient, ranging from 4.0% (w/v) polyacrylamide, at the top, to 24% polyacrylamide at the bottom (Emes et 01., 1975). In this case, we prefer to carry out the preliminary eleetrofocusing by the method of Doerr and Chrambach (1971) with a gel concentration of
459
3.5%T at 4 % C. The advantage of this technique is that virtually all protein material passes out of the gel rod and into the gel block during electrophoresis. Examples of the patterns obtained with normal sera, by each electrophoresis technique, are shown in Fig. I. The protein zones have been identified by appropriate staining and usually immunologically (Dewar and Latner, 1970; Emes et 01., 1975), using Ouchterlony double-diffusion against specific antisera. Taking into account such differences as haptoglobin types, it is remarkable how reproducible these serum protein patterns have proved in apparently healthy individuals. Protein-binding assay of cyclic AMP in plasma
Plasma cyclic AMP content has been determined by a radio-saturation technique using a specific binding protein from bovine adrenal cortices (Latner and Prudhoe, 1973). This competitive protein-binding method does not require preliminary extraction from plasma.
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Fig. I.-Patteras obtalDed wltb Dormal serum wbea gel IsoeIectrk: focuslaa was followed (A) by gel block e1ectrophoresis aad (B) by gel gradJeat electrophoresis. The proteID zoaes a,e beea staJaed wltb COOIDIIIIie brUUaat blue G. I, IgA; 2, laG; 3, 19M; 4, as-macroglobulla; 5, baptQlloblas; 6, CI-UpoproteID; 7, c:eruIopIumIa; II. lraasferrln; 9, haemopexla; 10, albumla; II, CI.-aatltrypsla; 12, CI.-acld glycoprotela; 13, prealbumla.
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460 A. L. Latner isoENZYME STUDIES IN CANCER
Serum lactate dehydrogenase is frequently elevated in patients suffering from malignancy (Hill and Levi. 1954). This finding is by no means constant but it is known that cancerous tissues contain a somewhat higher level of the enzyme than do the corresponding normal tissues (Meister. 19S0). This has led to the study of lactate dehydrogenase isoenzymes in malignant tumours. which have been found to contain mainly the slower moving isoenzymes LD-3. LD-4. and LD-S, and with certain exceptions this finding is irrespective of the tissue from which the tumour has been derived (Pfleiderer and Wachsmuth, 1961; Bllr et al., 1963; Richterich et al., 1963; Goldman et al., 1964; Latner et al., 1966; PoznanskaLinde et aI., 1966). The work in our own laboratory has been concerned to a large extent with tumours of the cervix, although we have ofcourse investigated
many other malignant tumours. The pattern obtained with extracts of cancer of the cervix compared with normal cervical mucosa is shown in Fig. 2. This work has been of great interest since we have shown that the malignancy pattern can also be found in preinvasive carcinoma in this situation. In fact with preinvasive carcinoma of the cervix there is a tendency for the pattern of the whole mucosa to be altered in the direction of malignancy, but the most marked change occurs at the site of the microscopically demonstrable lesion. This overall change supported the concept that cancer of the cervix could result from infection with a virus. which is now considered to be herpes virus type 2. Our studies with this aspect of preinvasive carcinoma were greatly helped by virtue of the development of a quantitative method for estimating isoenzymes of lactate dehydrogenase by reflectance densitometry (Latner and Turner. 1967).
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Wellcome award lecture 461 We have also found other isoenzyme changes in relation to cervical carcinoma. It is a matter of some great interest that the starch gel electrophoretic mobility of phosphogluconate dehydrogenase can vary from tumour to tumour. It was possible that this finding was conditioned by slight variations in experimental technique. This criticism has been overcome by determining. at the same time. the distribution of glucose 6-phosphate dehydrogenase. and as can be seen in Fig. 3 this enzyme showed no variation in mobility from tumour to tumour. It must be emphasised that these results were obtained from a slice of the same gel in which the variation of mobility of the other enzyme had been demonstrated. In view of the fact that it was known that certain viruses are associated with the production of malignant tumours in animals, and possibly in humans, we proceeded to see whether infection with such a virus, adenovirus 12, would change the isoenzyme pattern of lactate dehydrogenase of cells in tissue culture towards the pattern which has been shown to be associated with malignancy. We infected cultures of cynomolgus monkey kidney cells as well as cultures of human thyroid cells with adenovirus 12 or with poliomyelitis virus, which is not oncogenic. Only the adenovirus produced the expected changes. Of course, one of these viruses contains DNA and the other RNA. I doubt whether this difference is of great significance in the light of what we know about reverse transcriptase, Studies of LD isoenzyrnes have also been carried out on sera of patients suffering from cancer. Unfortunately the typical cancer pattern has not been found, probably owing to the different clear-
Fig. 4.-Lactate deIa~ ..tt.- obtaJMd after ClI'IU calt1n of _ _ IddIIey cortes. TIle riPt ..tten II tbe coatroI, the middle..tten WIll obtaJDed after treatIDeDt with calf d1r- blltoee ... tile left ..tten after treabDellt with rat IIYer blltoee. TIle arrow lIIIoWi the dJftcdoII oIl11Oft111e11t tonnll the . . . .
ance rates from the circulation of the different isoenzymes of lactate dehydrogenase. Unusual LD isoenzyme bands have occasionally been found in the tumours as well as in sera of patients suffering from cancer (Latner, 1964). One interesting finding was the high incidence of cancer in a family containing a newly described genetic variant. Others have described lactate dehydrogenase variants in one patient with chronic lymphocytic leukaemia and another with lymphoblastic sarcoma (Vesell, I96S). An additional LD-2 has been found in extracts of brain tumours (Soetens et al., 19M). It is interesting that the heat-resistant placental form of alkaline phosphatase occurs in the sera of a small proportion of patients suffering from various cancers, particularly carcinoma of the bronchus. This is known as the Regan isoenzyme (Fishman et 01.• 1968). HISTONE STUDIES
For many years, the concept has been held that the nuclear basic proteins, the so-called histones, are possibly concerned with gene control mechanisms. Since lactate dehydrogenase isoenzymes are built up from two sub-units (H and M), and there is a specific gene for each of these sub-units, it seemed highly worthwhile to investigate whether the addition of histones to tissues or cells in culture could affect the activity of either or both of these genes. This could be relatively easily done by studying any possible changes in the proportion of each of the five isoenzymes. In the first place (Latner and Longstaff, 19(9) observations were carried out on organ cultures of 1 mm l cubes of the cortex of the kidneys ofmice (Bar Harbor strain 129). Eight such cubes were kept alive in an appropriate organ culture vessel, which was a modification of one previously described (Dickson and Leslie, 1966). These portions of kidney cortex were actually placed on a Millipore filter inside a filter weD and the medium employed was the maintenance medium 199 (Burroughs Wellcome type TC22) containing the appropriate antibiotics to prevent bacterial growth. The isoenzyme patterns obtained with and without the histone are shown in Fig. 4. Compared with the normal control cubes the patterns showed interesting changes. When rat liver histone was employed, the spectrum of distribution of the isoenzymes altered in such a way that there was a preponderance of the slower moving forms. On the other hand when calf thymus histone was employed the change was in the opposite direction. Since liver contains more of the slower moving isoenzymes and thymus more of the faster moving entities, it could be concluded that there had been some kind of
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462
A. L. Latner
modification of gene activity in the direction of that in the organ from which the histone was derived. Although this change was quite obvious to the naked eye, reflectance densitometry and subsequent calculation of the absolute amounts of Hand M subunits per unit DNA indicated, at a satisfactory level of statistical probability, that rat liver histone had increased the M subunits and calf thymus histone the H subunits. Portions of kidney cortex, however, are made up of a number of different tissues, and this could complicate the interpretation of the results. It was therefore decided to test the effect of the histone preparations on tissue cultures. The cultures employed were BHKjC13 (baby hamster kidney cells) and Detroit 98 (human bone marrow cells). These were grown to confluence in Eagle's growth medium and then maintained in medium 199, with or without histone, for a period of 3 days. An extract of the culture of Detroit 98 cells now showed a pattern of all five LD isoenzymes, but LD-1 was present only in trace amounts and LD-3 and LD-4 were the predominant components. An extract of the BHKj C13 culture showed only LD-5. After histone treatment the isoenzyme patterns of the Detroit 98 cells behaved in a manner exactly similar to the organ cultures of mouse kidney cortex. The BHK/ C13 cells, however, which produce only the slowest moving form of lactate dehydrogenase, showed an increase of total lactate dehydrogenase activity after treatment with rat liver histone, the isoenzyme pattern sLiII showed only LD-5. The cells could not produce other isoenzymes after treatment with thymus histone. Observation with the microscope of the BHK/C13 rat liver histone treated culture showed very interesting changes. Normally, confluent cultures of this cell type show an ordered type of growth including the phenomenon of contact inhibition, resulting in the production of regular whorls of cells. After treatment with rat liver histone contact inhibition was lost, and some of the cells underwent changes in size and nuclear shape and, in fact, even became multinucleate. This kind of change is a manifestation of cell transformation and was another indication that histone treatment had greatly affected gene activity in a general sort of way. It is also interesting that when nuclear size in control cultures was compared with that in rat liver histone treated cultures there was a statistically significant increase in size of the nuclei after histone treatment (Latner et al., 1973). In other words there seems to have been an unfolding of the chromatin which is now believed to be consistent with an increase in general gene activity. The nuclear size was determined by staining a film of cells prepared under controlled
conditions on a glass slide in a Cytocentrifuge (Shandon). Using a projection microscope, at a fixed magnification, an image of the stained film was focused on to a sheet of Whatman 3MM chromatography paper. The projected images of nuclei could then be outlined in pencil and the shapes so obtained cut out and weighed. Each of these weights was, of course, related to the size of the original nucleus projected. One very interesting finding in relation to the nuclear size observations has been the discovery that not all histones are effective in this respect. Histone H4 is far and away the most active, whereas H2B and H3 have no effect at all. It could be argued that histones do not enter the cell cytoplasm or its nucleus, but produce their effect through some action on the cell membrane. It has been shown that histones can affect the electrophoretic mobility of cells (Latner and Turner, 1974), which is a property determined by the cell membrane. In order to show that histones do cross into the cell in tissue culture, we have prepared 1251 labelled histone and observed the cell uptake in two ways. Firstly, by fractionating the cell constituents (Hancock, 1974), it was possible to show that more of the histone was taken up by the nucleus than by the cytoplasm. Secondly, autoradiography showed quite clearly that labelled material had entered both the cytoplasm and the nucleus. The experiments with nuclear size and with the labelled material were carried out after exposure for an hour or so and the rapid take-up and action of histone is quite impressive. The findings would support the possible existence of a specific mechanism for nuclear uptake of histone. A study of the BHK/C13 and liver histone transformed cultures showed very clearly that many of the cells had characteristics usually associated with malignancy, and it was felt very worthwhile to follow up this concept. In the first place this was done by means of organ culture. Cells were grown to confluence in a filter well in the apparatus previously used for organ culture using Eagle's growth medium. The medium was then removed and replaced by maintenance medium with or without histone. Eight 1.0 mm s portions of mouse kidney cortex were placed on the confluent sheet of cells, the appopriate gas mixture was introduced, and the whole incubated for a further week. The cubes of kidney cortex were now sectioned and examined under the microscope. It was quite clear that invasion of the mouse kidney cortex by BHKjC13 cells had occurred only in the presence of histone. Similar invasion was obtained with polyoma-transformed cells, known to be malignant. It seemed, therefore, that we had possibly
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Wellcome award lecture 463 developed an in-vitro technique for demonstrating malignant invasion. Having demonstrated invasion in vitro, it was obviously necessary to see whether rat liver histone treated baby hamster kidney cells would produce malignant tumours in animals. With this in mind such cells were injected subcutaneously into the ftank region of hamsters. In each case, a tumour was produced which proved to be highly invasive locally and which nearly always produced generalised metastases. It has been possible to culture from one of these primary tumours a cell line which we have designated TRES ("tumour rescued") which, in spite of frequent subculturing, continues to produce fibrosarcomas in hamsters. Such cell lines are not too common and we have been particularly fortunate in being able to produce one. ANTI-PROTEINASE STUDIES
One very interesting finding with the in-vitro observations was that mouse kidney cells near the
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invading baby hamster kidney cells showed signs of lysis. This raised the possibility that invading cells were producing a proteinase. We therefore repeated the experiment using histone-containing maintenance medium. Half the flasks contained mouse kidney cortex cubes obtained in the usual way from our stock of mice. The remaining half contained the kidney cortex cubes obtained from mice injected with aprotinin (Trasylol) one hour before sacrifice. It is known that this aprotinin becomes concentrated in the kidney. These latter cubes of tissue therefore contained the anti-proteinase. It is very interesting to observe that there was inhibition of invasion by the baby hamster kidney cells of the mouse kidney cubes which contained the anti-proteinase. Since aprotinin prevented in-vitro invasion, we decided to try its effects in relation to the established TRES tumours. In animals which had been injected with aprotinin there was significant inhibition of growth as well as prevention of metastases. Just in case this effect was due to a specific property of the TRES cells we repeated the experiment using
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464 A. L. Latner
tumours(SMTC3H/HeVl)derivedfromspontaneous virgin female Heston C3H mouse. Results obtained were very similar to those with the TRES tumours. It seemed. therefore. that aprotinin inhibited growth of malignant tumours in animals and in fact caused them to undergo necrosis with spontaneous cure in a number of cases. Although this effect might seem to be related to the suggestion that malignant tumours obtain at least some of their nutrition by digesting the normal cells in their vicinity. further experiments by us have shown that the major effect of aprotinin is probably due to an enhancement of the activity of the immunological system (Latner and Turner. 1976). I am happy to state that we have obtained very similar results in a small group of humans. The group is not yet large enough for us to draw finn conclusions. As is well known. a potent anti-proteinase. alantitrypsin. is present in human blood. Because of
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findings with aprotinin, it seemed appropriate to study serum aI-antitrypsin in a group of patients suffering from some form of cancer. In the first place. we have chosen carcinoma of the cervix. It is a matter of some interest that the serum aI-antitrypsin as determined by the method of Mancini et al. (965) is raised both in the preinvasive and fully developed stages. In fact. the maximum rise has already been achieved when the tumour is still in the preinvasive stage. It could well be that the increased level of aI-antitrypsin is in some way related to enhancement of immunological responses and that immunological surveillance in relation to cancer could well depend. at least to some extent. upon this particular anti-proteinase. OUI
IsoELECTRIC FOCUSING FOLLOWED BY ELECTROPHORESIS
Since the technique is capable of demonstrating visually a number of immunoglobulins. viz 19A. laG. and IsM. when using gradient electrophoresis. it was obvious that it could well be used in the study of myelomatosis. This has been carried out quite effectively and different patterns successfully obtained in relation to the different types of this condition. The pattern of lsA myelomatosis. seen in Fi,. 5• shows a well-marked series of lsA zones but little or none of the IsG or IgM zones. Similarly. with laG myelomatosis there is a well-marked spot in the laG area. but the rest of this zone is missina alonl with lsA and IgM. We have also successfully studied conditions in which IgM is raised. and this immunoglobulin has been well demonstrated in the serum of patients suffering from Waldenstrom's macroglobulinaemia. It is also possible to demonstrate the presence of Bence-Jones protein in the blood. in the urine. and even in the cerebrospinal fluid. We have not yet had the opportunity of following the progress of the myelomatosis pattern in the individual patient. There is no doubt that this could and will be done. The technique has been extended to the study of proteins extracted from malisnant tumours and comparison of the patterns obtained with those from the tissue of origin of the tumour concerned. It is a matter of great interest that unusual protein spots have been found in cancers. These spots are not present in normal adult tissues. but can often be detected in fetal material. TheY do not necessarily correspond to careino-embryonic antigen or aIfetoprotein. Their situation would seem to indicate that there are undoubtedly a number of so-called cancer-specific proteins yet to be described. A typical example of such additional proteins can be seen in Fig. 6. which shows a pattern from a renal carcinoma.
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Wellcome award lecture STUDIES WITH CYCLIC
AMP
As already mentioned, in collaboration with Dr. Prudhoe a radiosaturation technique has been developed for the estimation of plasma cyclic AMP which can, of course, also be applied to urine and other biological fluids. These cyclic AMP studies have been extended to the sera of a group of patients suffering from cancer of the cervix, either preinvasive or fully established. Both cancer groups showed lower levels of cyclic AMP than did the normal controls matched for age. It was shown (Latner et al., 1975) that stage of the menstrual cycle or the use of oral contraceptives did not affect the plasma cyclic AMP level although a previous report had suggested that urinary excretion of this substance varied during the menstrual cycle. CONCLUSION
While I have dwelt on the development of new methodology, I hope, in conclusion, that I have demonstrated that the technique of application of such methods is the most rewarding. Especially is this the case in relation to medical problems. It is mainly for this reason that I believe that the education of all clinical biochemists should include at least some of the broad principles of pathology and a period of training in the wards. REFERENCES
Bar, U., Schmidt, E., Schmidt, F. W. Enzym-Muster und Isoenzyme menschlicher Tumoren. K/in. Wschr. 41 (1963) 977. Boyde, T. R. C., Latner, A. L. Starch-gel electrophoresis of transaminases in human tissue extracts and sera. Biochem. J. 82 (1962) 51. Coxon, R. V., Latner, A. L., King, E. J. Measurement of the concentration of Miracil in biological fluids. Trans. roy. Soc. trop, Med. Hyg. 41 (1947) 133. Dale, G., Latner, A. L. Isoelectric focusing in polyacrylamide gels. Lancet 1 (1968) 847. Dale, G., Latner, A. L. Isoelectric focusing of serum proteins in acrylamide gels followed by electrophoresis. C/in. chim. Acta 24 (1969) 61. Dewar, J. H., Latner, A. L. Immunological technique for identifying protein areas in gel slabs. C/in. chim. Acta 28 (1970) 149. Dickson J. A., Leslie, 1. The composition and metabolism of cells cultured in a modified filter-well apparatus. Exp. Cell Res. 41 (1966) 502. Doerr, P., Chrambach, A. Anti-estradiol antibodies: Isoelectric focusing in polyacrylamide gel. Analyt, Biochem. 42 (1971) 96. Ernes, A. V., Gallimore, M. J., Hodson, A. W., Latner, A. L. The preparation of crystalline human L-lactatenicotinamide-adenine dinucleotide oxidoreductase isoenzyme 1, involving preparative polyacrylamide-gel electrophoresis. Biochem. J. 143 (1974) 453.
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Emes, A. V., Latner, A. L., Martin, J. A. Electrofocusing followed bygradient electrophoresis: A two dimensional polyacrylamide gel technique for the separation of proteins and its applications to the immunoglobins. C/in. chim. Acta 64 (1975) 69. Fishman, W. H., Inglis, N. R .. Green. S., Anstiss, C. L., Ghosh, N. K., Reif, A. E., Rustigian, R., Krant, M. J., Stolbach, L. L. Immunology and biochemistry of regan isoenzyme of alkaline phosphatase in human cancer. Nature (Lond.) 219 (1968) 697. Goldman, R. D., Kaplan, N. 0., Hall, T. C. Lactic dehydrogenase in human neoplastic tissues. Cancer Res. 24 (1964) 389. Hancock, R. Interphase chromosomal deoxyribonucleoprotein..isolated as a discrete structure from cultured cells. J. molec. Bioi. 86 (1974) 649. Hill, B. R., Levi, C. Elevation of serum component in neoplastic disease. Cancer Res. 14 (1954) 513. Hodson, A. W., Latner, A. L., Raine, L., Skillen, A. W. Isoenzymes of human alkaline phosphatase and lactic dehydrogenase. J. Physiol, 159 (1961) 54. Hodson, A. W., Latner, A. L., Raine, L. Isoenzymes of alkaline phosphatase. Clin. chim. Acta 7 (1962) 255. Hodson, A. W., Latner, A. L. Alkaline phosphatase isoenzyrnes of human kidney and urine. C/in. chim. Acta 61 (1975) 53. Horn, D. B., Latner, A. L. The estimation of magnesium by atomic absorption spectrophotometry. C/in. chim. Acta 8 (1963) 974. Latner, A. L. A Colorimetric Method for the estimation of ergothioneine in human blood. Biochem. J. 42 (1948) xxxv. Latner, A. L., Pendlenton, G. B. A simple emergency method for estimation of blood sugar. Brit. med, J. 2 (1949) 418. Latner, A. L., Hodson, A. W. Isoenzymes of alkaline phosphatase. Proc, Ass. c/in. Biochem, 1 (1961) 149. Latner A. L., Skillen, A. W. Clinical applications of dehydrogenase isoenzyrnes. Lancet 2 (1961) 1286. Latner, A. L., Skillen, A. W. Visual Demonstration of Isoenzyme Activity after Starch-gel Electrophoresis. Proc. Ass. c/in. Biochem, 2 (1962) 3. Latner, A. L. Some clinical experiences with isoenzymes, Proc. Ass. c/in. Biochem., 3 (1964) 12. Latner, A. L. Phosphatase isoenzymes, in Enzymes in Clinical Chemistry, Ghent, 1964. Proceedings IVth West-European Symposium on Clinical Chemistry, p. 110. Amsterdam, Elsevier Publishing Company (1965). Latner, A. L., Turner, D. M., Way, S. A. Enzyme and isoenzyme studies in preinvasive carcinoma of the cervix. Lancet 2 (1966) 814. Latner, A. L., Turner, D. M. Quantitative Assay of Lactate Dehydrogenase Isoenzymes by Rellectance Densitometry. Clin. chim. Acta 15 (1967) 97. Latner, A. L., Skillen, A. W. Isoenzymes in Biology and Medicine. New York, Academic Press (1968). Latner, A. L., Longstaff, E. Modifications by crude histones of gene activity for lactate dehydrogenase. Nature (Land.) 224 (1969) 71. Latner, A. L., Parsons, M. E., Skillen, A. W. Isoelectric focusing of human liver alkaline phosphatase. Biochem. J. 118 (1970a) 299.
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A. L. Latner
Latner, A. L., Parsons, M. E., Skillen, A. W. Isoelectric focusing of alkaline phosphatase from human kidney and calf intestine. Enzymologia 40 (1970b) No.1. Latner, A. L., Longstaff, E., Lunn, J. M. Influence of histones on nuclear size. Nature New Biology 243 (1973) 107. Latner, A. L., Prudhoe, K. A simplified competitive protein binding assay for adenosine 3', 5' monophosphate in plasma. Clin, chim. Acta 48 (1973) 353. Latner, A. L., Turner, G. A. Surface modification and electrophoresis of normal and transformed BHK21 cells. J. Cell Sci. 14 (1974) 203. Latner, A. L., Patterson, S. W., Turner, G. A. Plasma levels of cyclic adenosine 3' 5' monophosphate in women with cancer of the cervix. IRCS med. Sci. 3 No.9 (1975) 459. Latner, A. L., Turner, G. A. Effect of aprotinin on immunological resistance in tumour-bearing animals. Brit. J. Cancer 30 (1976) 60. Mancini, G., Carbonara, A., Heremans, J. Immunochemical quantitation of antigens by single radial immunodiffusion. Int. J. Immunochem. 2 (1965) 235.
Meister, A. Lactic dehydrogenase activity of certain tumours and normal tissues. J. nat. Cancer Inst, 10 (1950) 1263. Pfleiderer, G., Wachsmuth, E. D. Age and function dependent differentiation of lactic dehydrogenase of human organs. Biochem. Z. 334 (1961) 185 (Ger.), Poznanska-Linde, H., Wilkinson, J. H., Withycombe, W. A. Lactate dehydrogenase isoenzymes in malignant tissues. Nature (Lond.) 209 (1966) 727. Richterich, R., Burger, A. Lactic dehydrogenase isoenzymes in human cancer cells and malignant effusions. Enzym, Bioi. CUn. (Basel) 3 (1963) 65. Smithies, O. An improved procedure for starch-gel electrophoresis: Further variations in the serum proteins of normal individuals. Biochem. J. 11 (1959) 585. Soetens, A., Karcher, D., Van Sande, M., Louenthal, A. Presence of additional lactate dehydrogenase isoenzymes in two cases of brain tumour. Enzymes in Clin. Chem. 4 (1965) 130. Vesell, E. S. Polymorphism of human lactate dehydrogenase isoenzyme. Science 148 (1965) 1103.
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Wellcome Award Lecture
A. L.
LATNER
University Department of Clinical Biochemistry, Royal Victoria Infirmary, Newcastle upon Tyne, NEl 4LP
It is indeed a great honour to receive the Wellcome
Latner and Hodson, 1961; Hodson et al., 1962; award, and I do this in all humbleness knowing quite Latner, 1965; Latner et al., 1970a, 1970b; Hodson well that much of my work has depended on colla- and Latner, 1975), as well as with the arninotransboration with a number of younger colleagues. In ferases (Boyde and Latner, 1962). Human LD-l has particular I wish to mention Dr. A. W. Skillen, been isolated in pure form as microcrystals (Emes Dr. G. A. Turner, Dr. Gordon Dale, Dr. A. V. Emes, et al., 1974) and I am now able to announce the and Mr. A. W. Hodson, although there have been isolation, also in pure form, of human liver alkaline phosphatase (Hodson and Latner, unpublished quite a number of other collaborators. The Wellcome award is given particularly for work observations). This was done by us more than a year related to the quality of laboratory practice. This, of ago. course, involves the development of new techniques, I first developed the concept that isoelectric and I should like to emphasise that technical focusing could be carried out in small gel rods at a development can be of two types. In the first, the time when I was watching a salesman's demonstrapurely analytical, there is the development of a tion of the large column in which focusing is done in technique of estimation, manual or automatic, which liquid. It occurred to me that a gel rod technique can be the means of estimating a substance, analysis would be not only more rapid but considerably more for which has not been previously available, or economical. This idea led to the co-operative work alternatively can improve such things as specificity, with Dr. Dale which culminated in the first publicaprecision, accuracy, cost, speed of determination, tion in the world literature of the technique of isoand the like. This type of development can in itself electric focusing in acrylamide gel (Dale and Latner, be a source of great satisfaction and a great help 1968). for the routine diagnostic laboratory. In this My studies with isoenzymes, isoelectric focusing, respect, I have had the good fortune to be involved and saturation assay using radioisotopes, I am happy in a number of instances. These have included the to say, have opened up a number of interesting estimation in body fluids of the therapeutic sub- fields. For the purpose of this lecture, however, I stance Miracil (Coxon et al., 1947), of ergothioneine have decided to discuss their relationship only to in human blood (Latner, 1948), a simple emergency cancer research carried out in my department. method for the estimation of blood glucose (Latner and Pendlenton, 1949) and the estimation of magneTECHNICAL METHODS sium by atomic absorption spectrophotometry (Horn and Latner, 1963), and, like most of us, an lsoenzymes even larger number of unpublished contributions. The second type of technical development is the Separation of isoenzymes is effected by vertical deliberate elaboration of methods intended solely starch-gel electrophoresis (Smithies, 1959) in starch for the purpose of investigation of a variety of blocks, 30 ern long, 0.6 ern thick, and of varying disease processes, not only in relation to diagnosis width, depending upon the number of samples to be but also to mechanisms of development of the examined. The samples are either sera or aqueous disease process itself. I personally have obtained extracts of tumours or normal tissues. The buffer even greater pleasure from this situation. Isoenzyme employed is either 0.05 M Tris-HCI, pH 8.6 (Latner studies, isoelectric focusing, and saturation assay and Skillen, 1961), or 0.025 M Tris-borate-EDTA at helve proved particularly rewarding in this respect. the same pH. A voltage gradient of 6 V/cm is Lactate dehydrogenase has been the isoenzyme applied for 2 hours. On completion of electrosystem mostly studied, by means of starch gel phoresis the gel is sliced into five slices, each of electrophoresis, using a modified Smithies's method which can be stained in a different way. To demon(1959) and a specific staining technique for enzyme strate lactate dehydrogenase (LD) isoenzyme bands activity (Latner and Skillen, 1962; Latner and a tetrazolium salt (MTT) is used, together with Turner, 1967; Latner and Skillen, 1968). Success phenazine methosulphate, NAD, and sodium has also been achieved with the isoenzymes of lactate in 0.3 M Tris-HCl buffer, pH 7.4. After human alkaline phosphatase (Hodson et al., 1961; incubation at 37°C the isoenzyme zones appear as 458
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Wellcome award lecture
purple bands due to formazan formation. Other dehydrogenase isoenzymes can be demonstrated in a similar manner by using the appropriate substrate (Latner and Skillen, 1968).
lsoelectric focusing followed by electrophoresis Both these procedures are carried out inacrylamide gels. Isoclectric focusing has been performed in an acrylamide gel rod, using Ampholine to produce the pH gradient. When focusing is complete, the gel rod is placed on top of a buffered gel block, into which the focused proteins are made to travel at pH 8.6 by means of electrophoresis. They can then be stained by an appropriate stain (Dale and Latner, 1969). We now prefer to use Coomassie brilliant blue G. The electrophoresis block can be a gradient, ranging from 4.0% (w/v) polyacrylamide, at the top, to 24% polyacrylamide at the bottom (Emes et 01., 1975). In this case, we prefer to carry out the preliminary eleetrofocusing by the method of Doerr and Chrambach (1971) with a gel concentration of
459
3.5%T at 4 % C. The advantage of this technique is that virtually all protein material passes out of the gel rod and into the gel block during electrophoresis. Examples of the patterns obtained with normal sera, by each electrophoresis technique, are shown in Fig. I. The protein zones have been identified by appropriate staining and usually immunologically (Dewar and Latner, 1970; Emes et 01., 1975), using Ouchterlony double-diffusion against specific antisera. Taking into account such differences as haptoglobin types, it is remarkable how reproducible these serum protein patterns have proved in apparently healthy individuals. Protein-binding assay of cyclic AMP in plasma
Plasma cyclic AMP content has been determined by a radio-saturation technique using a specific binding protein from bovine adrenal cortices (Latner and Prudhoe, 1973). This competitive protein-binding method does not require preliminary extraction from plasma.
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460 A. L. Latner isoENZYME STUDIES IN CANCER
Serum lactate dehydrogenase is frequently elevated in patients suffering from malignancy (Hill and Levi. 1954). This finding is by no means constant but it is known that cancerous tissues contain a somewhat higher level of the enzyme than do the corresponding normal tissues (Meister. 19S0). This has led to the study of lactate dehydrogenase isoenzymes in malignant tumours. which have been found to contain mainly the slower moving isoenzymes LD-3. LD-4. and LD-S, and with certain exceptions this finding is irrespective of the tissue from which the tumour has been derived (Pfleiderer and Wachsmuth, 1961; Bllr et al., 1963; Richterich et al., 1963; Goldman et al., 1964; Latner et al., 1966; PoznanskaLinde et aI., 1966). The work in our own laboratory has been concerned to a large extent with tumours of the cervix, although we have ofcourse investigated
many other malignant tumours. The pattern obtained with extracts of cancer of the cervix compared with normal cervical mucosa is shown in Fig. 2. This work has been of great interest since we have shown that the malignancy pattern can also be found in preinvasive carcinoma in this situation. In fact with preinvasive carcinoma of the cervix there is a tendency for the pattern of the whole mucosa to be altered in the direction of malignancy, but the most marked change occurs at the site of the microscopically demonstrable lesion. This overall change supported the concept that cancer of the cervix could result from infection with a virus. which is now considered to be herpes virus type 2. Our studies with this aspect of preinvasive carcinoma were greatly helped by virtue of the development of a quantitative method for estimating isoenzymes of lactate dehydrogenase by reflectance densitometry (Latner and Turner. 1967).
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Wellcome award lecture 461 We have also found other isoenzyme changes in relation to cervical carcinoma. It is a matter of some great interest that the starch gel electrophoretic mobility of phosphogluconate dehydrogenase can vary from tumour to tumour. It was possible that this finding was conditioned by slight variations in experimental technique. This criticism has been overcome by determining. at the same time. the distribution of glucose 6-phosphate dehydrogenase. and as can be seen in Fig. 3 this enzyme showed no variation in mobility from tumour to tumour. It must be emphasised that these results were obtained from a slice of the same gel in which the variation of mobility of the other enzyme had been demonstrated. In view of the fact that it was known that certain viruses are associated with the production of malignant tumours in animals, and possibly in humans, we proceeded to see whether infection with such a virus, adenovirus 12, would change the isoenzyme pattern of lactate dehydrogenase of cells in tissue culture towards the pattern which has been shown to be associated with malignancy. We infected cultures of cynomolgus monkey kidney cells as well as cultures of human thyroid cells with adenovirus 12 or with poliomyelitis virus, which is not oncogenic. Only the adenovirus produced the expected changes. Of course, one of these viruses contains DNA and the other RNA. I doubt whether this difference is of great significance in the light of what we know about reverse transcriptase, Studies of LD isoenzyrnes have also been carried out on sera of patients suffering from cancer. Unfortunately the typical cancer pattern has not been found, probably owing to the different clear-
Fig. 4.-Lactate deIa~ ..tt.- obtaJMd after ClI'IU calt1n of _ _ IddIIey cortes. TIle riPt ..tten II tbe coatroI, the middle..tten WIll obtaJDed after treatIDeDt with calf d1r- blltoee ... tile left ..tten after treabDellt with rat IIYer blltoee. TIle arrow lIIIoWi the dJftcdoII oIl11Oft111e11t tonnll the . . . .
ance rates from the circulation of the different isoenzymes of lactate dehydrogenase. Unusual LD isoenzyme bands have occasionally been found in the tumours as well as in sera of patients suffering from cancer (Latner, 1964). One interesting finding was the high incidence of cancer in a family containing a newly described genetic variant. Others have described lactate dehydrogenase variants in one patient with chronic lymphocytic leukaemia and another with lymphoblastic sarcoma (Vesell, I96S). An additional LD-2 has been found in extracts of brain tumours (Soetens et al., 19M). It is interesting that the heat-resistant placental form of alkaline phosphatase occurs in the sera of a small proportion of patients suffering from various cancers, particularly carcinoma of the bronchus. This is known as the Regan isoenzyme (Fishman et 01.• 1968). HISTONE STUDIES
For many years, the concept has been held that the nuclear basic proteins, the so-called histones, are possibly concerned with gene control mechanisms. Since lactate dehydrogenase isoenzymes are built up from two sub-units (H and M), and there is a specific gene for each of these sub-units, it seemed highly worthwhile to investigate whether the addition of histones to tissues or cells in culture could affect the activity of either or both of these genes. This could be relatively easily done by studying any possible changes in the proportion of each of the five isoenzymes. In the first place (Latner and Longstaff, 19(9) observations were carried out on organ cultures of 1 mm l cubes of the cortex of the kidneys ofmice (Bar Harbor strain 129). Eight such cubes were kept alive in an appropriate organ culture vessel, which was a modification of one previously described (Dickson and Leslie, 1966). These portions of kidney cortex were actually placed on a Millipore filter inside a filter weD and the medium employed was the maintenance medium 199 (Burroughs Wellcome type TC22) containing the appropriate antibiotics to prevent bacterial growth. The isoenzyme patterns obtained with and without the histone are shown in Fig. 4. Compared with the normal control cubes the patterns showed interesting changes. When rat liver histone was employed, the spectrum of distribution of the isoenzymes altered in such a way that there was a preponderance of the slower moving forms. On the other hand when calf thymus histone was employed the change was in the opposite direction. Since liver contains more of the slower moving isoenzymes and thymus more of the faster moving entities, it could be concluded that there had been some kind of
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462
A. L. Latner
modification of gene activity in the direction of that in the organ from which the histone was derived. Although this change was quite obvious to the naked eye, reflectance densitometry and subsequent calculation of the absolute amounts of Hand M subunits per unit DNA indicated, at a satisfactory level of statistical probability, that rat liver histone had increased the M subunits and calf thymus histone the H subunits. Portions of kidney cortex, however, are made up of a number of different tissues, and this could complicate the interpretation of the results. It was therefore decided to test the effect of the histone preparations on tissue cultures. The cultures employed were BHKjC13 (baby hamster kidney cells) and Detroit 98 (human bone marrow cells). These were grown to confluence in Eagle's growth medium and then maintained in medium 199, with or without histone, for a period of 3 days. An extract of the culture of Detroit 98 cells now showed a pattern of all five LD isoenzymes, but LD-1 was present only in trace amounts and LD-3 and LD-4 were the predominant components. An extract of the BHKj C13 culture showed only LD-5. After histone treatment the isoenzyme patterns of the Detroit 98 cells behaved in a manner exactly similar to the organ cultures of mouse kidney cortex. The BHK/ C13 cells, however, which produce only the slowest moving form of lactate dehydrogenase, showed an increase of total lactate dehydrogenase activity after treatment with rat liver histone, the isoenzyme pattern sLiII showed only LD-5. The cells could not produce other isoenzymes after treatment with thymus histone. Observation with the microscope of the BHK/C13 rat liver histone treated culture showed very interesting changes. Normally, confluent cultures of this cell type show an ordered type of growth including the phenomenon of contact inhibition, resulting in the production of regular whorls of cells. After treatment with rat liver histone contact inhibition was lost, and some of the cells underwent changes in size and nuclear shape and, in fact, even became multinucleate. This kind of change is a manifestation of cell transformation and was another indication that histone treatment had greatly affected gene activity in a general sort of way. It is also interesting that when nuclear size in control cultures was compared with that in rat liver histone treated cultures there was a statistically significant increase in size of the nuclei after histone treatment (Latner et al., 1973). In other words there seems to have been an unfolding of the chromatin which is now believed to be consistent with an increase in general gene activity. The nuclear size was determined by staining a film of cells prepared under controlled
conditions on a glass slide in a Cytocentrifuge (Shandon). Using a projection microscope, at a fixed magnification, an image of the stained film was focused on to a sheet of Whatman 3MM chromatography paper. The projected images of nuclei could then be outlined in pencil and the shapes so obtained cut out and weighed. Each of these weights was, of course, related to the size of the original nucleus projected. One very interesting finding in relation to the nuclear size observations has been the discovery that not all histones are effective in this respect. Histone H4 is far and away the most active, whereas H2B and H3 have no effect at all. It could be argued that histones do not enter the cell cytoplasm or its nucleus, but produce their effect through some action on the cell membrane. It has been shown that histones can affect the electrophoretic mobility of cells (Latner and Turner, 1974), which is a property determined by the cell membrane. In order to show that histones do cross into the cell in tissue culture, we have prepared 1251 labelled histone and observed the cell uptake in two ways. Firstly, by fractionating the cell constituents (Hancock, 1974), it was possible to show that more of the histone was taken up by the nucleus than by the cytoplasm. Secondly, autoradiography showed quite clearly that labelled material had entered both the cytoplasm and the nucleus. The experiments with nuclear size and with the labelled material were carried out after exposure for an hour or so and the rapid take-up and action of histone is quite impressive. The findings would support the possible existence of a specific mechanism for nuclear uptake of histone. A study of the BHK/C13 and liver histone transformed cultures showed very clearly that many of the cells had characteristics usually associated with malignancy, and it was felt very worthwhile to follow up this concept. In the first place this was done by means of organ culture. Cells were grown to confluence in a filter well in the apparatus previously used for organ culture using Eagle's growth medium. The medium was then removed and replaced by maintenance medium with or without histone. Eight 1.0 mm s portions of mouse kidney cortex were placed on the confluent sheet of cells, the appopriate gas mixture was introduced, and the whole incubated for a further week. The cubes of kidney cortex were now sectioned and examined under the microscope. It was quite clear that invasion of the mouse kidney cortex by BHKjC13 cells had occurred only in the presence of histone. Similar invasion was obtained with polyoma-transformed cells, known to be malignant. It seemed, therefore, that we had possibly
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Wellcome award lecture 463 developed an in-vitro technique for demonstrating malignant invasion. Having demonstrated invasion in vitro, it was obviously necessary to see whether rat liver histone treated baby hamster kidney cells would produce malignant tumours in animals. With this in mind such cells were injected subcutaneously into the ftank region of hamsters. In each case, a tumour was produced which proved to be highly invasive locally and which nearly always produced generalised metastases. It has been possible to culture from one of these primary tumours a cell line which we have designated TRES ("tumour rescued") which, in spite of frequent subculturing, continues to produce fibrosarcomas in hamsters. Such cell lines are not too common and we have been particularly fortunate in being able to produce one. ANTI-PROTEINASE STUDIES
One very interesting finding with the in-vitro observations was that mouse kidney cells near the
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invading baby hamster kidney cells showed signs of lysis. This raised the possibility that invading cells were producing a proteinase. We therefore repeated the experiment using histone-containing maintenance medium. Half the flasks contained mouse kidney cortex cubes obtained in the usual way from our stock of mice. The remaining half contained the kidney cortex cubes obtained from mice injected with aprotinin (Trasylol) one hour before sacrifice. It is known that this aprotinin becomes concentrated in the kidney. These latter cubes of tissue therefore contained the anti-proteinase. It is very interesting to observe that there was inhibition of invasion by the baby hamster kidney cells of the mouse kidney cubes which contained the anti-proteinase. Since aprotinin prevented in-vitro invasion, we decided to try its effects in relation to the established TRES tumours. In animals which had been injected with aprotinin there was significant inhibition of growth as well as prevention of metastases. Just in case this effect was due to a specific property of the TRES cells we repeated the experiment using
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464 A. L. Latner
tumours(SMTC3H/HeVl)derivedfromspontaneous virgin female Heston C3H mouse. Results obtained were very similar to those with the TRES tumours. It seemed. therefore. that aprotinin inhibited growth of malignant tumours in animals and in fact caused them to undergo necrosis with spontaneous cure in a number of cases. Although this effect might seem to be related to the suggestion that malignant tumours obtain at least some of their nutrition by digesting the normal cells in their vicinity. further experiments by us have shown that the major effect of aprotinin is probably due to an enhancement of the activity of the immunological system (Latner and Turner. 1976). I am happy to state that we have obtained very similar results in a small group of humans. The group is not yet large enough for us to draw finn conclusions. As is well known. a potent anti-proteinase. alantitrypsin. is present in human blood. Because of
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findings with aprotinin, it seemed appropriate to study serum aI-antitrypsin in a group of patients suffering from some form of cancer. In the first place. we have chosen carcinoma of the cervix. It is a matter of some interest that the serum aI-antitrypsin as determined by the method of Mancini et al. (965) is raised both in the preinvasive and fully developed stages. In fact. the maximum rise has already been achieved when the tumour is still in the preinvasive stage. It could well be that the increased level of aI-antitrypsin is in some way related to enhancement of immunological responses and that immunological surveillance in relation to cancer could well depend. at least to some extent. upon this particular anti-proteinase. OUI
IsoELECTRIC FOCUSING FOLLOWED BY ELECTROPHORESIS
Since the technique is capable of demonstrating visually a number of immunoglobulins. viz 19A. laG. and IsM. when using gradient electrophoresis. it was obvious that it could well be used in the study of myelomatosis. This has been carried out quite effectively and different patterns successfully obtained in relation to the different types of this condition. The pattern of lsA myelomatosis. seen in Fi,. 5• shows a well-marked series of lsA zones but little or none of the IsG or IgM zones. Similarly. with laG myelomatosis there is a well-marked spot in the laG area. but the rest of this zone is missina alonl with lsA and IgM. We have also successfully studied conditions in which IgM is raised. and this immunoglobulin has been well demonstrated in the serum of patients suffering from Waldenstrom's macroglobulinaemia. It is also possible to demonstrate the presence of Bence-Jones protein in the blood. in the urine. and even in the cerebrospinal fluid. We have not yet had the opportunity of following the progress of the myelomatosis pattern in the individual patient. There is no doubt that this could and will be done. The technique has been extended to the study of proteins extracted from malisnant tumours and comparison of the patterns obtained with those from the tissue of origin of the tumour concerned. It is a matter of great interest that unusual protein spots have been found in cancers. These spots are not present in normal adult tissues. but can often be detected in fetal material. TheY do not necessarily correspond to careino-embryonic antigen or aIfetoprotein. Their situation would seem to indicate that there are undoubtedly a number of so-called cancer-specific proteins yet to be described. A typical example of such additional proteins can be seen in Fig. 6. which shows a pattern from a renal carcinoma.
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Wellcome award lecture STUDIES WITH CYCLIC
AMP
As already mentioned, in collaboration with Dr. Prudhoe a radiosaturation technique has been developed for the estimation of plasma cyclic AMP which can, of course, also be applied to urine and other biological fluids. These cyclic AMP studies have been extended to the sera of a group of patients suffering from cancer of the cervix, either preinvasive or fully established. Both cancer groups showed lower levels of cyclic AMP than did the normal controls matched for age. It was shown (Latner et al., 1975) that stage of the menstrual cycle or the use of oral contraceptives did not affect the plasma cyclic AMP level although a previous report had suggested that urinary excretion of this substance varied during the menstrual cycle. CONCLUSION
While I have dwelt on the development of new methodology, I hope, in conclusion, that I have demonstrated that the technique of application of such methods is the most rewarding. Especially is this the case in relation to medical problems. It is mainly for this reason that I believe that the education of all clinical biochemists should include at least some of the broad principles of pathology and a period of training in the wards. REFERENCES
Bar, U., Schmidt, E., Schmidt, F. W. Enzym-Muster und Isoenzyme menschlicher Tumoren. K/in. Wschr. 41 (1963) 977. Boyde, T. R. C., Latner, A. L. Starch-gel electrophoresis of transaminases in human tissue extracts and sera. Biochem. J. 82 (1962) 51. Coxon, R. V., Latner, A. L., King, E. J. Measurement of the concentration of Miracil in biological fluids. Trans. roy. Soc. trop, Med. Hyg. 41 (1947) 133. Dale, G., Latner, A. L. Isoelectric focusing in polyacrylamide gels. Lancet 1 (1968) 847. Dale, G., Latner, A. L. Isoelectric focusing of serum proteins in acrylamide gels followed by electrophoresis. C/in. chim. Acta 24 (1969) 61. Dewar, J. H., Latner, A. L. Immunological technique for identifying protein areas in gel slabs. C/in. chim. Acta 28 (1970) 149. Dickson J. A., Leslie, 1. The composition and metabolism of cells cultured in a modified filter-well apparatus. Exp. Cell Res. 41 (1966) 502. Doerr, P., Chrambach, A. Anti-estradiol antibodies: Isoelectric focusing in polyacrylamide gel. Analyt, Biochem. 42 (1971) 96. Ernes, A. V., Gallimore, M. J., Hodson, A. W., Latner, A. L. The preparation of crystalline human L-lactatenicotinamide-adenine dinucleotide oxidoreductase isoenzyme 1, involving preparative polyacrylamide-gel electrophoresis. Biochem. J. 143 (1974) 453.
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Emes, A. V., Latner, A. L., Martin, J. A. Electrofocusing followed bygradient electrophoresis: A two dimensional polyacrylamide gel technique for the separation of proteins and its applications to the immunoglobins. C/in. chim. Acta 64 (1975) 69. Fishman, W. H., Inglis, N. R .. Green. S., Anstiss, C. L., Ghosh, N. K., Reif, A. E., Rustigian, R., Krant, M. J., Stolbach, L. L. Immunology and biochemistry of regan isoenzyme of alkaline phosphatase in human cancer. Nature (Lond.) 219 (1968) 697. Goldman, R. D., Kaplan, N. 0., Hall, T. C. Lactic dehydrogenase in human neoplastic tissues. Cancer Res. 24 (1964) 389. Hancock, R. Interphase chromosomal deoxyribonucleoprotein..isolated as a discrete structure from cultured cells. J. molec. Bioi. 86 (1974) 649. Hill, B. R., Levi, C. Elevation of serum component in neoplastic disease. Cancer Res. 14 (1954) 513. Hodson, A. W., Latner, A. L., Raine, L., Skillen, A. W. Isoenzymes of human alkaline phosphatase and lactic dehydrogenase. J. Physiol, 159 (1961) 54. Hodson, A. W., Latner, A. L., Raine, L. Isoenzymes of alkaline phosphatase. Clin. chim. Acta 7 (1962) 255. Hodson, A. W., Latner, A. L. Alkaline phosphatase isoenzyrnes of human kidney and urine. C/in. chim. Acta 61 (1975) 53. Horn, D. B., Latner, A. L. The estimation of magnesium by atomic absorption spectrophotometry. C/in. chim. Acta 8 (1963) 974. Latner, A. L. A Colorimetric Method for the estimation of ergothioneine in human blood. Biochem. J. 42 (1948) xxxv. Latner, A. L., Pendlenton, G. B. A simple emergency method for estimation of blood sugar. Brit. med, J. 2 (1949) 418. Latner, A. L., Hodson, A. W. Isoenzymes of alkaline phosphatase. Proc, Ass. c/in. Biochem, 1 (1961) 149. Latner A. L., Skillen, A. W. Clinical applications of dehydrogenase isoenzyrnes. Lancet 2 (1961) 1286. Latner, A. L., Skillen, A. W. Visual Demonstration of Isoenzyme Activity after Starch-gel Electrophoresis. Proc. Ass. c/in. Biochem, 2 (1962) 3. Latner, A. L. Some clinical experiences with isoenzymes, Proc. Ass. c/in. Biochem., 3 (1964) 12. Latner, A. L. Phosphatase isoenzymes, in Enzymes in Clinical Chemistry, Ghent, 1964. Proceedings IVth West-European Symposium on Clinical Chemistry, p. 110. Amsterdam, Elsevier Publishing Company (1965). Latner, A. L., Turner, D. M., Way, S. A. Enzyme and isoenzyme studies in preinvasive carcinoma of the cervix. Lancet 2 (1966) 814. Latner, A. L., Turner, D. M. Quantitative Assay of Lactate Dehydrogenase Isoenzymes by Rellectance Densitometry. Clin. chim. Acta 15 (1967) 97. Latner, A. L., Skillen, A. W. Isoenzymes in Biology and Medicine. New York, Academic Press (1968). Latner, A. L., Longstaff, E. Modifications by crude histones of gene activity for lactate dehydrogenase. Nature (Land.) 224 (1969) 71. Latner, A. L., Parsons, M. E., Skillen, A. W. Isoelectric focusing of human liver alkaline phosphatase. Biochem. J. 118 (1970a) 299.
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Latner, A. L., Parsons, M. E., Skillen, A. W. Isoelectric focusing of alkaline phosphatase from human kidney and calf intestine. Enzymologia 40 (1970b) No.1. Latner, A. L., Longstaff, E., Lunn, J. M. Influence of histones on nuclear size. Nature New Biology 243 (1973) 107. Latner, A. L., Prudhoe, K. A simplified competitive protein binding assay for adenosine 3', 5' monophosphate in plasma. Clin, chim. Acta 48 (1973) 353. Latner, A. L., Turner, G. A. Surface modification and electrophoresis of normal and transformed BHK21 cells. J. Cell Sci. 14 (1974) 203. Latner, A. L., Patterson, S. W., Turner, G. A. Plasma levels of cyclic adenosine 3' 5' monophosphate in women with cancer of the cervix. IRCS med. Sci. 3 No.9 (1975) 459. Latner, A. L., Turner, G. A. Effect of aprotinin on immunological resistance in tumour-bearing animals. Brit. J. Cancer 30 (1976) 60. Mancini, G., Carbonara, A., Heremans, J. Immunochemical quantitation of antigens by single radial immunodiffusion. Int. J. Immunochem. 2 (1965) 235.
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