UPTAKE OF [ 8 H]URIDINE INTO PRECURSOR POOLS AND RNA IN OSTEOGENIC CELLS

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1 J. Cell Set. 2, (1967) Printed in Great Britain UPTAKE OF [ 8 H]URIDINE INTO PRECURSOR POOLS AND RNA IN OSTEOGENIC CELLS MAUREEN OWEN Medical Research Council, Bone-Seeking Isotopes Research Unit, The Churchill Hospital, Oxford SUMMARY Young rabbits were given a single intraperitoneal injection of fhjuridine. Using the technique of water-soluble autoradiography a study was made of the uptake of the radioactive label into soluble precursors and RNA in cells on an actively growing bone surface. Labelling of the soluble intracellular pools was immediate, but incorporation of label from these pools into RNA was not completed until 24 h after injection. At this time all the label in the sections was in RNA but this represented only 30 % of the total label initially in the soluble pools. This means that 70 % of the label is lost from the cell in the first 24 h either as degradation products of RNA synthesis or by other as yet unknown mechanisms. The pattern of labelling of the RNA was similar to that previously found for other mammalian cells in vivo or in vitro. There was a rapid uptake of label into nuclear RNA which reached a maximum by 2 h after injection and a slower uptake into cytoplasmic RNA which reached a maximum by 24 h after injection. There was a slow loss of label from the cells after 24 h indicating a half-life of about 8 days for this relatively stable RNA. A comparison was made of RNA synthesis in the proliferating preosteoblasts and the highly differentiated nondividing osteoblasts. Labelling of the nuclear RNA for the two cell types was identical. The rate of labelling of the cytoplasmic RNA was similar for the two cell types but the maximum level of labelling in the cytoplasm of the osteoblasts was 2 to 3 times that in the preosteoblasts. This could be correlated with the more active protein synthesis by the osteoblasts. There was a slow loss of labelled RNA by the osteoblasts and preosteoblasts and a rapid loss by the osteocytes after the cells had been incorporated within the bone. It was suggested that this loss paralleled the decline in the rate of protein synthesis by the cells as their environment changed. INTRODUCTION The osteogenic cells on the growing periosteal surface of the mid-shaft of a long bone include several stages of cellular differentiation, analogous to that found in most growing tissues. The fully differentiated cells, the osteoblasts, are engaged in the production of the organic bone matrix, and form a single layer lining the bone surface. The precursors of these cells, here called the preosteoblasts, form a layer behind the osteoblasts, and are the main region of cell division. (Preosteoblasts were first so-called by Pritchard (1952). They are probably equivalent to cells in similar situations which have been referred to by other names: spindle and reticulum cells (Heller, McClean & Bloom, 1950), mesenchymal cells (Kember, i960; McClean & Urist, 1961); the cambium layer (McClean & Urist, 1961); and osteoprogenitor cells (Young, 1963).) The rate of cell proliferation in the preosteoblast layer is balanced by the loss of cells through differentiation to osteoblasts, and the increase of the total

2 4o M. Owen cell population in the process of bone growth. Behind the preosteoblasts is a layer of fibroblasts separating the osteogenic layers from nearby muscle (Fig. i). In previous work we have measured various parameters of this system, using as our experimental material the periosteal surface of the shaft of i- to 2-week-old rabbit femur. It was found that, in these young, actively growing animals, the preosteoblasts and osteoblasts spend on average 3 days on the bone surface before becoming enclosed in bone, either as cells within the haversian canals or as osteocytes embedded in the bone matrix. The bone surface advances, in the process of bone growth, at the Osteoblasts Fibroblasts Bone surface Calcified bone Fig. 1. Diagrammatic representation of the periosteal surface of the shaft of the femur of a rabbit aged 1 to 2 weeks, illustrating (a) the various layers of cells on the bone surface and (b) the position of the bone surface after 4 days' growth. rate of about 70 fi per day, and it was found that each osteoblast produces approximately its own volume of organic bone matrix per day during the time it spends on the bone surface (Owen, 1963). About 95 % of the organic matrix of bone consists of the protein, collagen, and the other 5 % of mucopolysaccharides and non-collagenous protein (Herring, 1964). Hence the osteoblasts are a typical example of highly differentiated cells engaged mainly in the synthesis of a few specific substances, and in particular collagen. We have, therefore, a system which includes different stages of cellular differentiation: the preosteoblasts, although a mixed population of cells, are engaged mainly in cell proliferation; the osteoblasts on the bone surface are cells in a state of maximum functional activity; and finally, there are the osteocytes and the 'haversian osteoblasts' which are a later and more quiescent stage in the process of differentiation. It seemed of interest therefore to investigate ribonucleic acid (RNA) synthesis and decay in this system, and particularly, (1) its pattern in the different cell types, (2)

3 Uptake of uridine in osteogenic cells 41 in relation to protein synthesis and (3) in relation to the different stages of cell differentiation. The present paper describes an autoradiographic study of RNA metabolism in this system following an injection of uridine-5-h 3. Very little work has been done, hitherto, on the pattern of RNA synthesis with time in vivo, and on the variation of this pattern in different cells of a mammalian tissue. What studies there are have usually been made (with some exceptions, e.g. Burckard, Fontaine & Mandel (1959) and Loeb, Howell & Tomkins (1965)) over short time periods, and have used biochemical cell-fractionation techniques. Since the latter inevitably involve mixed cell populations a comparison from one cell type to another has not been possible, nor can it be certain, using these techniques, that the cell population is not changing significantly during the period of the experiment. In the present study measurements have been made over a period of 10 days after injection. Grain counts of the cells in their relative positions in histological sections have enabled a comparison of the different cell types to be made. All the cells present at the time of injection are preserved during the period of the experiment, and it has been possible to follow the decay of labelled RNA in the same cells during both the time they are in their maximal functional state and as they pass from one stage of differentiation to another. In addition to the standard methods of preparing autoradiographs using fixed, paraffin-embedded sections, the technique of water-soluble autoradiography has also been used. With this method it has been possible to study the uptake of label into the soluble intracellular precursor pools and the rate of incorporation from these pools into RNA. It was found that uptake of the radioactive label from the pools continued for about 24 h after injection. In all cell types studied the uptake of labelled precursor into RNA was initially very rapid into the nucleus, and slower into the cytoplasm, in agreement with the results found so far for all cells in vivo or in vitro (for a review see Prescott, 1964). Some interesting differences between the different cell types are discussed in detail. MATERIALS AND METHODS Dutch rabbits between 1 and 2 weeks of age were chosen according to weight, g. For the autoradiographic studies, uridine-5-h 3, specific activity about 20 c/mm, obtained from the Radiochemical Centre, Amersham, was used as an RNA precursor. An intraperitoneal injection of 5/*c/g was given and the rabbits killed at intervals varying from 15 min to 10 days after injection. A small number of rabbits was used for the purpose of determining the level of uridine in the serum after an intraperitoneal injection. For this experiment uridine-2- C 14 (Amersham) was injected and the animals killed at intervals up to a few hours after injection. The blood was collected and the serum separated by centrifugation. A known quantity of serum was dried on a planchet and counted using a scintillation counter. The counts were compared with those from a known volume of the injection solution and the results were expressed as a percentage of the injected dose.

4 42 M. Owen Autoradiographs (i) Frozen sections, soluble components retained. The method of making autoradiographs of frozen sections retaining the soluble components has been developed and described in detail by Appleton (1964). Small pieces of the mid-shaft of the femur were taken straight from the animal and frozen as rapidly as possible by plunging into liquid nitrogen, 196 C. This could usually be accomplished within 3 min. The frozen tissue was then sealed in plastic-aluminium foil and stored in a liquid-nitrogen refrigerator until required. Coverslips were coated with AR 10 stripping film with the emulsion side upwards, dried and brought down to Cryostat temperatures of 20 C. Sections of the frozen tissue 5 fi thick were cut on the Cryostat at 20 C and these were picked up on the film on the coverslips at approximately the same temperature. This was done in a dark room using a Kodak Wratten No. 1 safelight at a distance of 6 ft. The coverslip with the film and the section was then placed in a pre-cooled black plastic box and stored in a deep freeze at 20 C for autoradiographic exposure, usually 5 days. After exposure the sections were fixed at 17 C for 1 min in formalacetic-alcohol, a mixture consisting of 40% formaldehyde, glacial acetic acid and absolute ethanol in the proportions 10:3:87. After washing they were develbped for 5 min in D19 B, rinsed in distilled water and fixed for 10 min in Johnson's Fixsol, diluted 1: io, all at 17 C. After further washing the sections were stained with methyl green and pyronin and the whole coverslip preparation was then inverted and mounted on a slide (Appleton, 1964). The soluble components will, of course, have been washed out during washing, fixation and processing. This, however, does not matter since their image has already been obtained on the photographic film. (it) Frozen sections, extracted. Frozen sections were obtained as described above in (i). In this case the section was picked up on a pre-cooled slide. Sections were then extracted at o C, using various solutions, as will be described later. The preparation was thoroughly washed and brought to room temperature, and autoradiographs were prepared using AR 10 stripping film(pelc, 1956) as previously described (Owen, 1963). These were dried and then stored for autoradiographic exposure at 20 C. The exposure time was the same as in (t). After exposure the sections were fixed at 17 C for I min in formal-acetic-alcohol. These last steps were taken so that the procedures for obtaining autoradiographs with and without soluble components should be as parallel as possible. The autoradiographs were processed exactly as described in (i). A comparison of sections prepared using methods (t) and (u) could be made, and the amount of label removed during extraction determined. (Hi) Paraffin sections. The bones were fixed in 10% neutral buffered formalin for 3 days and decalcified in ethylenediaminetetra-acetic acid, ph 7, for 3 weeks. Small pieces of the mid-shaft of the femur were taken through the alcohols, cedarwood oil, benzene and finally embedded in paraffin wax. Sections 5 fi thick were placed on slides and autoradiographs prepared with AR 10 strippingfilm (Owen, 1963). Exposure times were days depending on the grain density being counted. A total of 33 rabbits from 6 litters was used for these autoradiographs. Sections from

5 Uptake of uridine in osteogenic cells 43 animals of the same litter were placed together on the same microscope slide and covered with a single piece of film. The results for any one litter were not therefore dependent on differences in the autoradiographic film or development procedure. Counting of autoradiographs. Counts were made of the number of grains per nucleus and per cytoplasm. The tritium /?-ray has a short range, its average value in tissue or photographic emulsion being less than 1 ji. At least two corrections must be made to the grain counts if one is to compare the total amounts of labelled material in the different cell structures. First, in autoradiographs of sections more than 90 % of the grains counted are those arising from a volume whose dimensions are the area of the nucleus or the cytoplasm which is in contact with the emulsion, by about 2 fi deep. This volume, which gives rise to grains, in the emulsion should be the same fraction of the total volume in each case if the 3 H-activity of the two structures is to be compared. It can easily be seen that the greater the cross-sectional area the smaller the fraction of the total volume represented. However, knowing the average crosssectional area of the nucleus and cytoplasm and assuming that the nucleus and the cell are spherical in shape the grain counts can be corrected so that they represent the relative activity in the total volume of the nucleus and cytoplasm. Taking the nucleus of the osteoblast as the reference, all other results were corrected accordingly. Secondly, a correction should also be made for the different self-absorption of the /?-rays in the nucleus and cytoplasm. In the case of liver cells the relative self-absorption of the nucleus and cytoplasm has been measured for tritium /?-rays (Maurer & Primbsch, 1964) and it was found that the grains over the cytoplasm must be increased by about 40 % relative to the nucleus, if the activity of the two is to be compared. This correction has not been made in the present study, since self-absorption depends on the dry mass per unit area of the structures concerned and these have not been measured for bone cells. Although it should be remembered that this correction is likely to be considerable, it does not, however, affect the conclusions drawn from the present work. All grain counts have been corrected for background grains which were usually between i-o and 1-5 grains per 100 /t 2. RESULTS Activity in the blood The level of activity in the plasma during the first hour after an intraperitoneal injection, expressed as a percentage of the injected dose, is plotted in Fig. 2. The level was only about 4% at 10 min after injection and had fallen to less than 1 % by 1 h. Autoradiographs A diagrammatic representation of the system studied, the periosteal surface of the shaft of the femur, is shown in Fig. 1. The bone surface has characteristic loops typical of forming haversian systems. The different cell types are indicated in Fig. 1 (a), and the situation after 4 days' growth is illustrated in Fig. 1 (b). Frozen sections. The results obtained from autoradiographs made using methods (i)

6 44 M. Owen and (it) are described below. Autoradiographs of frozen sections made using method (i) do not come into contact with water until after the autoradiographic exposure. A record of the amount and position of all radioactive material, water-soluble or otherwise, is thus retained in the autoradiograph. These are compared with autoradiographs made using method («) where the main difference is that the section has been extracted before the autoradiograph exposure. Some preliminary experiments using method (it) were performed with different extracting solutions for different periods of time. The solutions used were 5% trichloroacetic acid (TCA), 10% neutral formalin and formal-acetic-alcohol. The 10 Activity in serum o T3 I SO 60 Minutes Fig. 2. Graph showing the rate of fall-off of activity in the serum following a single intraperitoneal injection of uridine-2-c 14. frozen section, mounted on a slide, was extracted at 0 C for 1, 2, 3 or 4 min. The amount of radioactive label removed was the same for all times and for the 3 solutions used. The results described below are for TCA extraction for 2 min at o C. TCA extraction is commonly used in nucleic acid determinations (Hutchison & Munro, 1961) as a preliminary step to remove the acid-soluble small molecules such as free nucleotides, etc. The radioactively labelled, acid-insoluble material remaining is assumed to be in macromolecular RNA. Autoradiographs of a frozen section which has been extracted and of a frozen section which has not been extracted are compared

7 Uptake of undine in osteogenic cells 45 in Figs. 10 and 11 for an animal killed 1 h after a single intraperitoneal injection of [ 3 H]uridine. Note the grains throughout both the nucleus and the cytoplasm in the section which has not been extracted (Fig. 10). In Fig. u, the section was extracted for 2 min in 5 % TCA at o C C, the majority of the radioactive material has now been removed from the cytoplasm and what remains is almost entirely in the nucleus. In comparing Figs. 10 and 11, note that the exposure time for the former is 5 days and for the latter 8 days, so that the amount of label removed by extraction is greater than is apparent from a direct comparison of the two photographs. 100 c,total label in cytoplasm -Total label in nucleus 10 o -V 10 RNA nucleus V RNA cytoplasm i V 72 Hours Fig. 3. Graph showing grains per nucleus and grains per cytoplasm in osteoblasts with time after injection; solid curves are for non-extracted frozen sections with all soluble components retained, broken curves are for frozen sections extracted for 2 min at o C in 5 % TCA, exposure time 5 days. Counts were made of the grains per nucleus and per cytoplasm in osteoblasts in both extracted and unextracted sections, and the results are shown in Fig. 3. Each point is the average grain count of approximately 100 cells. The dotted lines show the label in frozen sections extracted with TCA or in other words in RNA. The label in the unextracted sections, which includes both soluble material and RNA, is shown by the solid curves in Fig. 3. As can be seen, at 1 h after injection only 10% of the total label is in RNA, the rest is in soluble form. By 8 h about 50 % of the label is in RNA, and by 24 h all the label is in RNA, a negligible amount remaining in the soluble pools at this time and thereafter. Furthermore, when all the label is out of the pools and is present solely in RNA it can be seen that this represents only about 30 % of the amount initially in the soluble pools. Using the results in Fig. 3 the average loss

8 46 M. Ovien per hour of soluble material from the pools and the average increase of RNA per hour can be calculated for different times after injection and the resulting curves are shown in Fig. 4. If the amount of label which appears in RNA per hour is expressed as a proportion of the soluble label which is lost per hour, it can be seen from Fig. 4 that this proportion increases with time after injection. Between 1 and 2 h after injection the amount of label which appears in RNA is equivalent to about 28 % of the soluble label which is lost, by 10 h to about 50%, and by 24 h practically all of the 10 p 10 a O J Hours Fig. 4. Graph showing the variation with time after a single intraperitoneal injection of ["Frjuridine of the rate of loss from the pool9 and the rate of increase of RNA. A, loss of soluble label per hour from pools (nuclear and cytoplasmic); B, increase in insoluble label (RNA) per hour in nucleus; C, increase in insoluble label (RNA) per hour in cytoplasm. These curves were obtained using the results in Fig. 3. soluble label is being converted into RNA. It will also be noted in Figs. 3 and 4 that there is a very rapid appearance of label in nuclear RNA and a much slower appearance in the cytoplasm. Paraffin sections. The results for grains per nucleus and per cytoplasm in osteoblasts, in autoradiographs of paraffin sections over the first 24 h are shown in Fig. 5. These curves are qualitatively identical with the results for the appearance of labelled RNA in the extracted sections, broken curves, Fig. 3. This permits some confidence in assuming that the label present in autoradiographs of paraffin sections is representative of RNA. We have therefore studied the synthesis and retention of RNA over an extended period of 10 days using autoradiographs of paraffin sections which are

9 Uptake of uridine in osteogenic cells p Osteoblasts, Cytoplasm e 10 o Nucleus Hours Fig. 5. Graph showing grains per nucleus and grains per cytoplasm in osteoblasts with time after injection, for paraffin sections, exposure time 10 days. Position of periosteal bone surface Ac time of injection,. 1 day after injection ' 2 days after injection 3 days after in/ection 4 days after injection 5 days after injection 6 days after injection 7 days after injection 8 days after injection 9 days after injection 10 days after injection Fig. 6. Diagram showing the position of the periosteal bone surface at different times up to 10 days after injection, and illustrating the areas counted at the different times (see text). technically easier to obtain. In the work with paraffin sections each point on the graphs is an average grain count of about 200 cells. Preosteoblasts and osteoblasts. Counts were made of the number of grains per nucleus and per cytoplasm in both preosteoblasts and osteoblaats over a period of 10 days after injection. Since the bone surface is continually advancing in the process of growth (Fig. 1) the cells on the surface of the bone are changing with time. A

10 48 M. Owen diagram of the areas of cells counted at different times after injection is shown in Fig. 6. The cells were counted in squares of side 67 fi which is about the distance advanced by the bone surface per day. These squares were designated A, B, C, etc., starting from the perio9teal surface inwards. With each succeeding day after injection an additional square further from the bone surface was included to take account of bone growth. In this way it was possible to follow the pattern of RNA synthesis and decay in the cells as their position and function in bone changes, as is described below. Up to and including 1 day after injection the cells in squares A and B were counted. For all practical purposes these represent the initially labelled population of cells on the bone surface. At later times, for example 4 days after injection, the cells, which were on the surface at one day, that is in A and B, are by now within bone, either as osteocytes or as cells in haversian canals. Since bone is more cellular on its surface than in its interior, it was estimated that the cells in squares B, C, D and E at 4 days are essentially equivalent to the population of cells which had been labelled during the first day on the surface in squares A and B (Fig. 6). Once the cells are within bone it is assumed that they keep their same relative position, so that at 6 days the cell population initially labelled on the bone surface is now represented by the cells in squares D, E, F and G, and at 8 days and 10 days by those in F, G, H and I, and H, I, J and K, respectively. It is the results for the cells in these particular squares at the respective times after injection which are given in Figs. 7 and 8. Where a cell is on the surface of a haversian canal it is included as an osteoblast; where it is within the canal but not on the surface it has been included as a preosteoblast. The maximum labelling of the RNA is attained by 24 h after injection, the time at which all the label is in an acid-insoluble form as demonstrated by the results from frozen sections. It will be noticed (Figs. 7, 8), that the same pattern of labelling with time is followed for both preosteoblasts and osteoblasts. The curves for the nuclear labelling are almost identical for the two cell types. The nuclear labelling quickly reaches its maximum by about 2 h, stays approximately constant at this level until about 24 h and then falls off slowly. In the case of the cytoplasm the curves have a similar shape for the two cells. The label appears there more slowly than in the nucleus, rises to a maximum at 24 h and then falls off slowly. In the preosteoblasts, however, the maximum level of labelling reached in the cytoplasm is much lower than in the osteoblasts. Osteocytes. There are two situations in which the osteocytes were seen to be labelled. In the first case, a few hours after injection, the newest osteocytes near to the periosteal bone surface in the squares A and B (Fig. 6) were labelled. At 2 h the average level of labelling in these cells was about one-third of the labelling of the adjacent osteoblasts on the bone surface. Osteocytes more distant from the bone surface were not significantly labelled immediately after injection even at the longest autoradiographic exposure times. In the second case, labelled osteocytes were also seen at later times after injection by virtue of the fact that the osteoblasts, which were initially labelled on the bone surface, eventually become incorporated either as osteocytes in bone matrix or as osteoblasts on the surface of a haversian canal (Fig. 1). These labelled osteocytes are in no danger of being confused with those in the first category since

11 Uptake of uridine in osteogenic cells Osteoblasts Cytoplasm g 1-0 O 0-1 I I I Days Fig. 7. Grains per nucleus and grains per cytoplasm in osteoblasts up to 10 days after injection measured on autoradiographs of paraffin sections, exposure time 10 days. 10 r Preosteoblasts Cytoplasm 0 'g Li Days Fig. 8. Grains per nucleus and grains per cytoplasm in preosteoblasts up to 10 days after injection measured on autoradiographs of paraffin sections, exposure time 10 days. 4 Cell Sci. 2

12 50 M. Owen they are embedded in the bone matrix at a later time and therefore in a different position. For example at 3 and 4 days after injection, the osteocytes in A (Fig. 6), that is the most newly formed osteocytes, are cells which were osteoblasts about 1 day previously. At the time of their incorporation into the matrix the osteocytes in A will therefore have a degree of labelling approximately the same as the labelling of the osteoblasts in B in the same section. Similarly the labelling of osteocytes in B and C can be compared with the labelling of the osteoblasts in C and D. The ratio of the grain count in osteocytes to the grain count in the corresponding osteoblasts has been plotted in Fig. 9 for 4 different animals, 3 killed at 3 days and 1 at 4 days after injection. Here the grain count in osteocytes in squares A, B and C has been compared with the grain count in the osteoblasts in squares B, C and D A A i O _ - O A A A B B C I c D Osteocytes- Osteoblasts Fig. 9. Ratio of grains per cell in osteocytes and osteoblasts of the same age, each symbol representing one animal; 3 of the animals were killed at 3 days after injection and 1 at 4 days (see text). respectively in the same section. These are all cells which have been incorporated within bone after the initial uridine injection. As was expected the labelling in the newest osteocytes, square A, is of the same order as that in the corresponding osteoblasts, square B. The results for the deeper squares, B and C, show that there is a rapid loss of RNA from the osteocytes after being embedded in matrix for several days, by comparison with the osteoblasts of about the same age which remain on the surfaces in the haversian canals. These cells in fact lose relatively little of their labelled RNA in the same period. The fibroblasts (Fig. 1) which surround the osteogenic layer of the periosteum have previously been found (Owen, 1963) to be a rather inert population of cells with a low rate of cell division. They also have a very low level of RNA synthesis. The level of labelling in the fibroblasts was less than one-tenth the level of labelling in the osteoblasts on a per cell basis at 2 h after injection. There was also a low level of labelling in the bone matrix, particularly near the growing surface, but a detailed study of this has not yet been made.

13 Uptake of uridine in osteogenic cells 51 DISCUSSION Intracellular precursor pools and RNA metabolism. There is good evidence to show that RNA precursors, such as labelled nucleosides, are first rapidly incorporated into intracellular precursor pools of small molecules. RNA synthesis then proceeds using these labelled intermediates and, depending upon the size and duration of the pool, the labelling may continue for some time after a pulse of labelled nucleoside has been given (Harris, 1959; Watts & Harris 1959; Graham & Rake, 1963; Bresnick, Lanclos & Gonzales, 1965). The importance of intracellular precursor pools has been emphasized by Harris and his colleagues, working with an in vitro system (Harris, 1959; Harris & Watts, 1962; Watts 1964a). They have illustrated the persistence of radioactive label in these pools for periods of hours following uptake of a labelled RNA precursor, and have shown that the pools cannot easily be diluted by transfer of the cells to a non-radioactive medium (Watts, 19646). The presence of these pools in vivo has been confirmed in the present work. The experiments with uridine-2-c 14 show that labelling of the pools occurs very rapidly. Less than 4% of the dose is left in the serum within 10 min after a single intraperitoneal injection and the level is still falling very steeply. A single injection can thus be looked upon as a 'flash' labelling of the intracellular RNA precursor pools. However, although labelling of the pools is very fast, uptake of label from these pools into RNA is not completed until about 24 h after injection. The duration of these acid-soluble precursor pools in osteoblasts in vivo is thus relatively long, radioactive label persisting in the pools for up to 24 h after injection. In an earlier experiment (Owen, 1966) we have shown that a 'chase' injection of non-radioactive uridine at 100-fold concentration, given 1 h after injection of radioactive uridine, did not affect the amount of label incorporated into RNA at later times, indicating that in vivo, dilution of the label in these pools also does not take place easily. The same result has been found in liver by Loeb et al. (1965), who showed that a chase of non-radioactive orotic acid given 4i h after the original injection of labelled acid did not significantly affect the uptake of labelled precursor into RNA. It does not appear to be possible, therefore, to dilute the radioactive label in these precursor pools by following with a chase of the same unlabelled precursor. In vitro, however, it has been shown that these pools can be diluted to some extent in certain circumstances. This was found to depend on the particular radioactive precursor used and on the type of non-labelled precursors added to the medium (Harris, 1963; Watts, 19646). Our results also show (Fig. 4) that the proportion of the soluble pool which is incorporated into RNA increases with time after injection. This implies that the form in which the radioactive material exists in the pools changes with time. This observation, together with the difficulty in diluting the radioactive label in the precursor pools, suggests that the pools themselves are probably a well-organized sequence of events leading up to RNA synthesis, and not a random supply of building-blocks available for RNA synthesis. In the present work it has been shown that there is a very rapid initial incorporation into nuclear RNA which reaches its maximum within a few hours and a slower 4-2

14 52 M. Owen appearance of labelled RNA in the cytoplasm. This result is in agreement with previous results for all mammalian cells which have been studied (Prescott, 1964) and with previous work on bone (Burckard et al. 1959; Young, 1963). In the results for osteoblasts obtained from frozen sections, it was shown that, by 24 h after injection when there is no longer any label in the precursor pools, the amount of label in RNA is only about 30 % of that initially in the pools. Consequently about 70% of the initially labelled soluble material does not end up in the more stable RNA which reaches its maximum by 24 h. The form in which this label is lost from the cell is not known. Some of it may be lost via reactions which are not involved in RNA synthesis and some in the form of degradation products of RNA synthesis, particularly the rapidly labelled nuclear RNA. It is known that there is considerable re-utilization and recycling of degradation products in RNA synthesis but a proportion of them is also lost from the cell (Harris, 1963, 1964a; Watts, 19646). Our results are in broad agreement with those from numerous studies on RNA metabolism in mammalian tissues following a pulse of labelled nucleoside or other precursor. At short times (within a few hours) after exposure to a labelled precursor, a rapidly labelled fraction of RNA has constantly been reported (Harris et al. 1963; Perry, 1964; Revel & Hiatt, 1964). It is synthesized in the nucleus and a large proportion of it appears to be degraded there (Harris, 19646). The function of this rapidly labelled RNA is not certain (Harris, 1964 a), though it has been suggested that it may be an important site of response to such influences as hormones (Hiatt et al. 1965). It has been characterized using sucrose density-gradient techniques (Perry, 1964), and has been found to be made up of polydisperse components. It is thought that this rapidly labelled RNA is made up of several fractions with different half-lives (Watts, 19646) and that it may include short-lived messenger RNAs, soluble RNAs and macromolecular precursor forms of ribosomal RNA (Perry, 1964; Perry, Srinivasan & Kelley, 1964; Hiatt et al. 1965) as well as other as yet unrecognized species. At longer times after injection (about a day) sedimentation analysis of the labelled RNA has shown that the bulk of the radioactive label becomes coincident with the 3 optical density peaks characteristic of the bulk of the cellular RNA, i.e. in large part ribosomal RNA (Perry, 1964; Revel & Hiatt, 1964; Loeb et al. 1965). Consequently, in our experiments the more stable RNA component, which reaches its maximum in the cytoplasm at 24 h after injection, is likely to consist mostly of ribosomal RNA. Some of it must also be present in the nucleus since the level of labelling there does not fall off after it has reached its maximum at 2 h, but remains high until 24 h and then falls off in parallel with the cytoplasm. In addition to ribosomal RNA this more stable RNA may also contain some messenger RNA since there is recent evidence that, in highly differentiated cells, there exists a fraction of messenger RNA with a long half-life which is as stable as the ribosomes themselves (Hiatt et al. 1965). Turnover of RNA It is of interest to know the amount of RNA turnover which occurs in these cells, where turnover is defined as a balanced process of synthesis and degradation of RNA. In the case of the rapidly labelled RNA which is synthesized early within the first 24 h

15 Uptake of uridine in osteogenic cells 53 after injection, it is not possible to make any estimate of true RNA turnover due to the presence of label in the precursor pools and to the likelihood of recycling of degradation products which return to the pools and are re-used in RNA synthesis (Harris, 1964a). In the case of the more stable RNA which reaches its maximum by 24 h after injection, an estimate of the turnover of this RNA can be made from the slope of the curve after one day (Figs. 7, 8). After this time the amount of label in the precursor pools is negligible and consequently re-utilization of degradation products, although it cannot be completely ruled out, is not likely to be very significant. Within the limits of accuracy of the experimental results, the curves after one day are exponential and indicate a half-life of about 8 days for this relatively stable component of RNA. The half-life of ribosomal RNA in rat liver, measured by a similar method, was found to be of the same order of magnitude, about 5 days (Loeb et al. 1965; Hiatt et al. 1965). Although the results are not very precise the slopes of the curves for the fall-off in RNA after one day appear to be the same in the osteoblast and preosteoblast and in both the nucleus and cytoplasm (Figs. 7, 8). To what can this slow loss of RNA be attributed? Some of it may be turnover in conjunction with protein synthesis. However, in view of the similar slopes of the curves for the different cell types it seems more likely that this loss of RNA runs in parallel with the fall-off in the rate of protein synthesis by the cells as they become incorporated within bone. For example, osteoblasts are most active during the time they spend on the bone surface; after they have become osteoblasts on the surfaces of haversian canals, their rate of production of matrix decreases (Owen, unpublished). Comparison of the different cell types It has been pointed out that the same pattern of labelling is followed both for preosteoblasts and osteoblasts, and similar results have also been found for osteoclasts in these animals (Bingham & Owen, unpublished). It is of interest that the curves for the nuclear labelling are practically identical for the two cell types. In the case of the cytoplasm the curves have a similar shape but the maximum level of labelling attained in the cytoplasm at one day is lower in the case of the preosteoblasts. It is likely that there is some correlation between the level of labelling of RNA in the cell cytoplasm and the amount of protein extruded by different cells. In the present work the osteoblasts certainly synthesize more protein than the preosteoblasts, if the uptake of radioactive glycine is any measure of protein synthesis in the two cell types. It has been found that on a unit-area basis the uptake of glycine by the osteoblasts is about 10 times that of the preosteoblasts in this system (Owen, unpublished). The fate of an osteoblast, which has been labelled whilst active on the bone surface, is to become either an osteocyte embedded in bone matrix or an osteoblast on the surface of a haversian canal (Fig. 1). Osteocytes and osteoblasts of the same age were compared, and it was found that whereas the osteoblast loses only a few per cent of its label the osteocyte loses about 70 % of its label, and presumably its RNA, within 2 days. Although osteocytes continue to synthesize protein for a short time after they have been enclosed in bone matrix (Young, 1962; Owen, 1963), the loss of RNA by

16 54 M. Owen the osteocyte cannot be explained by loss due to protein synthesis, since this effect would be too small. For example, in the case of the osteoblasts, even if the loss of RNA was entirely due to protein synthesis this is only a few per cent per day at a time (between i and 4 days) when they are synthesizing approximately their own volume of protein per day (Fig. 7). The osteocyte must synthesize less than its own volume in toto and yet in 2 days loses most of its label. This loss of label by osteocytes is very likely similar to the loss of label by osteoblasts and preosteoblasts after being enclosed within haversian canals, but the effect is much more dramatic in the case of the osteocytes. The rapid loss of RNA by the osteocyte can thus probably be correlated with the rapid decline in its rate of protein synthesis after being embedded in bone matrix. In the case of all 3 cell types, preosteoblasts, osteoblasts, and osteocytes, it is therefore suggested that the loss of RNA after one day parallels the decrease in metabolic activity of the cells as their environment changes. In conclusion, following an intraperitoneal injection of [ 3 H]uridine the intracellular pools of RNA precursors become labelled almost immediately. The radioactive label persists in these pools for a considerable period, uptake of the label into RNA not being completed until about 24 h after injection. One of the limitations of the present techniques is the fact that it is not possible to characterize the different RNAs synthesized. Recently, development of selective extraction methods indicates that this limitation may be overcome to some extent in the future (Woods & Zubay, 1965). Our results show that there is a rapid appearance of RNA first in the nucleus and a slower appearance in the cytoplasm. They do not, however, enable us to say whether any, and if so, how much, of this rapidly labelled nuclear RNA is a precursor of the cytoplasmic RNA. The fact that the curves for the nuclear RNA are practically identical for the two cell types in spite of their different metabolic activities, whereas the cytoplasmic curves are quantitatively very different, would suggest that the relationship between nuclear and cytoplasmic RNA is not a simple one. This compares with recent work on cells of the cerebral cortex in newborn and adult rats (Adams, 1966), where a similar type of result has been found. The loss of RNA by the osteocyte raises an interesting speculation concerning the recent interest in this cell as a target for the action of parathyroid hormone on bone (B61anger, 1965; Talmage et al. 1965). A possible mechanism of the hormone action on this cell would be via the initiation of protein (or enzyme) synthesis. This, of course, would involve initiation of RNA synthesis which could be detected using the present techniques. These effects are, at present, in the course of investigation. The author gratefully acknowledges the help and advice of Dr Janet Vaughan throughout this work. She would like to thank Professor H. Harris, Dr S. R. Pelc and Miss P. Bingham for reading the manuscript and making helpful suggestions. She is most grateful to Miss I. Brazell and Mrs V. Wilkinson for their expert technical assistance.

17 REFERENCES Uptake of uridine in osteogemc cells 55 ADAMS, D. H. (1966). The relationship between cellular nucleic acids in the developing rat cerebral cortex. Biochem. J. 98, 636-^640. APPLETON, T. C. (1964). Autoradiography of soluble labelled compounds, jfl R. microsc. Soc. 83, BALANCER, L. F. (1965). Osteolysis: an outlook on its mechanism and causation. In The Parathyroid Glands (ed. P. J. Gaillard, R. V. Talmage & A. M. Budy), pp University of Chicago Press. BRESNICK, E., LANCLOS, K. & GONZALES, E. (1965). The biosynthesis of ribonucleic acid in the liver of the rat fetus in vivo. Biockim. biophys. Acta 108, BURCKARD, J., FONTAINE, R. & MANDEL, P. (1959). Metabolisme des acides ribonucleiques de l'os de lapin et de rat in vivo. C. r. Sianc. Soc. Biol. 153, GRAHAM, A. F. & RAKE, A. V. (1963). RNA synthesis and turnover in mammalian cells propagated in vitro. A. Rev. Microbiol. 17, HARRIS, H. (1959). Turnover of nuclear and cytoplasmic ribonucleic acid in two types of animal cell, with some further observations on the nucleolus. Biochem. J. 73, HARRIS, H. (1963). Nuclear ribonucleic acid. In Progress in Nucleic Acid Research, vol. 2 (ed. J. N. Davidson & W. E. Cohn), pp New York: Academic Press. HARRIS, H. (1964a). Function of the short-lived ribonucleic acid in the cell nucleus. Nature, Land. 201, HARRIS, H. (19646). Transfer of radioactivity from nuclear to cytoplasmic ribonucleic acid. Nature, Lond. 202, HARRIS, H., FISHER, H. W., RODGERS, A. L., SPENCER, T. & WATTS, J. W. (1963). An examination of the ribonucleic acids in the Hela cell with special reference to current theory about the transfer of information from nucleus to cytoplasm. Proc. R. Soc. B 157, HARRIS, H. & WATTS, J. W. (1962). The relationship between nuclear and cytoplasmic ribonucleic acid. Proc. R. Soc. B 156, HHII.HR, M., MCCLEAN, F. C. & BLOOM, W. (1950). Cellular transformations in mammalian bones induced by parathyroid extract. Am. J. Anat. 87, HERRING, G. M. (1964). Chemistry of the bone matrix. Clin. Orthop. no. 36, pp HIATT, H. H., HENSHAW, E. C, HIRSCH, C. A., REVEL, M. & FINKEL, R. (1965). Interrelations of protein and ribonucleic acid metabolism in mammalian tissues. Israel J. med. Sci. 1, I323-I333- HUTCHISON, W. C. & MUNRO, H. N. (1961). The determination of nucleic acids in biological materials. Analyst, Lond. 86, KEMBER, N. F. (i960). Cell division in endochondral ossification. J.BoneJt Surg. 42B, LOEB, J. N., HOWELL, R. R. & TOMKINS, G. M. (1965). Turnover of ribosomal RNA in rat liver. Science, N.Y. 149, MAURER, W. & PRIMBSCH, E. (1964). Grosse der /?-Selbst-Absorption bei der 3 H-Autoradiographie. Expl Cell Res. 33, MCCLEAN, F. C. & URIST, M. R. (1961). Bone: An Introduction to the Physiology of Skeletal Tissue, 2nd edition. University of Chicago Press. OWEN, M. (1963). Cell population kinetics of an osteogenic tissue. I. J. Cell Biol. 19, OWEN, M. (1966). RNA synthesis in growing bone. Third European Symposium on Calcified Tissues (ed. H. Fleisch, H. J. Blackwood & M. Owen), pp PELC, S. R. (1956). The stripping film technique of autoradiography. Int. J. appl. Radiat. Isotopes 1, PERRY, R. P. (1964). Role of the nucleolus in ribonucleic acid metabolism and other cellular processes. Natn. Cancer Inst. Monogr. no. 14, PERRY, R. P., SRINIVASAN, P. R. & KELLEY, D. E. (1964). Hybridization of rapidly labelled nuclear ribonucleic acids. Science, N. Y. 145, PRESCOTT, D. M. (1964). Cellular sites of RNA synthesis. In Progress in Nucleic Acid Research and Molecular Biology, vol. 3 (ed. J. N. Davidson & W. E. Cohn), pp New York: Academic Press. PRITCHARD, J. J. (1952). A cytological and histochemical study of bone and cartilage formation in the rat. J. Anat. 86,

18 56 M. Owen REVEL, M. & HIATT, H. H. (1964). The stability of liver messenger RNA. Proc. natn. Acad. Sci. U.S.A. 51, TALMAGE, R. V., DOTY, S. B., COOPER, C. W., YATES, C. & NEUENSCHWANDER, J. (1965). Cytological and biochemical changes resulting from fluctuations in endogenous parathyroid hormone levels. In The Parathyroid Glands (ed. P. J. Gaillard, R. V. Talmage & A. M. Budy), pp University of Chicago Press. WATTS, J. W. (1964a). Turnover of nucleic acids in a multiplying animal cell. 1. Incorporation studies. Biochem.J. 93, WATTS, J. W. (19646). Turnover of nucleic acids in a multiplying animal cell. 2. Retention studies. Biochem.J. 93, WATTS, J. W. & HARRIS, H. (1959). Turnover of nucleic acids in a non-multiplying animal cell. Biochem.J. 72, WOODS, P. S. & ZUBAY, G. (1965). Biochemical and autoradiographic studies of different RNAs: Evidence that transfer RNA is chromosomal in origin. Proc. natn. Acad. Sci. U.S.A. 54, YOUNG, R. W. (1962). Autoradiographic studies on post natal growth of the skull in young rats injected with tritiated glycine. Anat. Rec. 143, YOUNG, R. W. (1963). Nucleic acids, protein synthesis and bone. Clin. Orthop. 26, {Received 9 September 1966) Figs. 10, 11. Autoradiographs of part of the periosteal surface of the shaft of a rabbit femur, aged 1-2 weeks (see Fig. 1) 1 h after a single intraperitoneal injection of [ 3 H]- uridine. Fig. 10. Frozen section with soluble components retained, exposure time 5 days. Fig. 11. Frozen section extracted for 2 min at o C in 5 % TCA, exposure time 8 days. Bone, b; fibroblast, /; osteoblast, ob; preosteoblast, pob.

19 Journal of Cell Science, Vol. z, No. i W^-.,fj u M. OWEN {Facing p. 56)

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