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Growth regulations in normal and leukaemic haemopoietic stem cells
Leukemia Resrarch, Vol. I, Nos. 213, pP. 153-156. Pergamon Preu, 1977. Printed in Great Britain.
GROWTH REGULATIONS IN N O R M A L A N D LEUKAEMIC HAEMOPOIETIC STEM CELLS L. G. LAJTHA, B. I. LORD,J. MoRI and E. WRIGHT Paterson Laboratory, Christie Hospital and Holt Radium Institute, Manchester, U.K. THE QUESTIONraised by this conference is whether leukaemic cells have any 'controlling' effects on normal stem cells (and on the normal baemopoiesis in general) and whether leukaemic cells obey----even in part--the normal regulatory mechanism of the haemopoietic system. The fact that in leukaemia not infrequently normal haemopoietic elements decrease in number has been known and in animal leukaemia it has been demonstrated ([1] Fig. 1). The question, of course, arises whether such effects are due to a propensity of the leukaemic cells to produce some form of normal control or whether these effects are due to 'toxic' products of such cells. Such toxic products, theoretically, may be specific products of the cells in which case their identification could be
of diagnostic and prognostic significance. On the other hand, they could be non-specific metabolic breakdown products in the leukaemic tissue which is known to carry a significant non growth fraction and concommitant cell death. As far as the normal controlling mechanisms are concerned we can speak at two levels of control, as far as our present understanding is concerned. The first refers to certain inhibitory control materials, particularly in the 'recognisible' maturing end cells (promyelocytes, myelocytes and early normoblasts). Such materials have been described by Ryt6maa and Kiviniemi [2] and their observations have been confirmed and extended by Lord et al. [3, 4] as illustrated in Table 1. This indicates that some low LCFU in BODY
I
FRACTION d [r~'~U -'l'c'~]
01
LCFU
in SPLEEN or MARROW
lo6
ms CU~
1.0~
""
J
._~
"~
""....
\~
IJ I
i
I
l
i
i
i
,
i
I
2
3
4
S
6
7
II
9
"
DAYS after IOs LEUK.SPLEEN CELLS i.v.
.ACUm ~. normal agar colony forming units in FIG. I. LCFU, leukaemic colony forming cells; .ACU, J marrow or spleen; Repop, repopulating capacity of marrow. 153
154
L. G. LAJ'rHA,B. I. LORD,J. Morn and E. Wgiotrr TABLE 1. SPECmcrrms OF ALL EXTRACTS Factors GCE LNE NBME
IV
CFUs
CFUc
PHA lymph
n gran
CML gran
'AML'
ALL
0 ?
0 0
0 +
+ 0
+ O
+?
0 0
+
-
LI210 P388 leukaemia leukaemia 0 0
0 0
GCE, granulocyte extract; LNE, lymph node extract; NBME IV, fraction IV from normal bone marrow (non cycling); 0, no effect; +, inhibition; n gran, normal granulocytes; CML gran, granulocytes from chronic myeloid leukaemia.
molecular weight products of these cells have a highly specific feedback control effect on the proliferation of the respective cell lines. Three points must be emphasised: firstly, that the controlling materials are non cytotoxic, their action is reversible and their action is not a shut down of proliferation but simply an elongation of the respective cell cycle times. We are therefore talking about cell line specific, cell cycle length modulation. Such modulation, of course, can have very powerful effect in any amplifying transit population such as the erythroid or granulopoietic system. Taking a more or less predetermined maturation time, the number of cell cycles during that maturation process will determine the degree of amplification. Since the maturation process is essentially a 'suicide' process whereby cells will reach a non dividing state, an extra division during that time (effected by, e.g. shortening of the cell cycle) will double the output, conversely one division less (by lengthening the cell cycle times) will halve the output. The high degree of specificity of these compounds is further indicated by our observation that neither of them have any effect on the proliferation of the stem cells or of the committed precursor cells. Very little is known about proliferation in the committed precursor cells (for the erythroid or granulopoietic lines) but recently a fair amount of information became available concerning proliferation control in the pluripotent stem cell population. That some local control is operating, has been indicated by the observation that pluripotent stem cells are not randomly distributed within the marrow cavity [5]. Shielding experiments of parts of the haemopoietic tissue have also indicated that proliferation control is of a local nature [6, 7], similar conclusions were drawn in phenylhydrazine (PHZ) treated animals by Rencricca et al. [8]. Recent experiments in our laboratory indicated that the factor responsible for such local stem cell proliferation control can be easily extracted from the cells. Mixing cells from proliferating or non proliferating haemopoietic tissues indicated that a
TABLE 2. EFFECTo F CYCLING OR NON CYCLING STEM CELLS ON STEM CELL PROLIFERATION IN MIXED CELL SUSPENSIONS
SHTdR kill PHZ PHZ PHZ PHZ
BM alone BM+irr PHZ Sp. Sp alone Sp+irr PHZ BM
~40% ~15~o ~
5 ~o
~35~o
Ratio of irradiated 'signal' cells to assay cells=2:l. The irradiation (900 rad) was performed to prevent colony formation by the 'signal' cells. TABLE 3.
STIMULATORY AND INI.flBrrORY FRACTIONS FOR STEM CELL PROLIFERATION IN NORMAL A N D REGENERATING BONE MARROW
CFUs 'kill' by SHTdR 'cycling' BM Control (no addition) N BME fr. III. N B M E f r . IV. R BME fr. III.
~ 35 ~o ~ 35 ~o -*~ 5~o ~35~
'non-cycling' BM ~ 5 ~ 5 ~ 5 -*~25~
R B M E fr. I V .
~ 35 ~o
~
fr. I. and II. NBME/RBME
~ 35 ~
,~ 5
5
NBME'~extracts from normal (non-cycling) and reRBMEJgenerating (cycling) bone marrow. ft. HI "~30-50,000 and 50--100,000 tool. wt. fractions. ft. IV j respectively. compound is released from the cells. If irradicated cells from non regenerating marrow, or spleen, are mixed with regenerating stem cells (regenerating marrow) a factor profoundly inhibits the cycling of the latter. Conversely from regenerating marrows a factor appears to be released which initiates cycling of resting stem cells (E. Wright, to be published): Table 2. With brief incubation of regenerating or normal bone marrow and Amicon filtration of their cell free supernates [9] we were able to demon-
Growth regulations in normal and leukaemic haemopoietic stem cells strate the presence of a stem cell specific inhibitor fraction and f r o m regenerating marrows a stimulating fraction; Table 3. The specificity of the inhibitor is indicated by the observation that while it can completely shut down the proliferation o f pluripotent stem cells, it will not effect the cycling of, e.g. a c o m m i t t e d granulopoietic precursor. These observations indicate a practical possibility of looking at various leukaemic cell populations. Both cell line specific cell cycle modulators and the stem cell specific inhibitor can now be isolated in reasonably clean state and their effect on leukaemic cells can be investigated. Clearly appropriate extracts can be made from the various leukaemias and the presence of such factors can be looked for. This is likely to give considerable additional information on the nature of the ieukaemic process. Finally I wish to recall the fact that in C M L the pluripotent stem cells seem to be functionally normal in most respects;
155
they seem to o b e y - a t least p a r t i a l l y - - n o r m a l proliferation control [10] (Fig. 2). It will be of particular interest to study how this property of the stem cell changes during the blastic transformation.
I
c,, Ph-
.{.~1 i~lii
Both Pit- and Ph I earn diffecentiate
r.,. Ph I i ~ p l e t e l ) ,
¢~b'olled
1
diff.
FIG. 2. Scheme of stem cell population with a Ph ~ clone in it.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
TANAKAT., TESTA N. E. G. & L~u'rI-IAL. G. (1973) Leukaemic stem cell kinetics in experimental animals unifying concepts of leukaemia. Bibhhca haemat. No. 39. RVT6MAA T. & KtV]NmMX K. (1968) Control of granulocyte production. I--Chalone and antichalone, l l - specific humoral regulators. Cell Tiss. Kinet. 1, 329. LORDB. I., C-~xcEK L., CEXCEK B., ShAH G. P., DEXTER T. M. & L~JTHA L. G. (1974) lnhibitors of haemo poietic cell proliferation?: specificity of action with the haemopoietic system. Br. J. Cancer 29, 168. LORD B. I., CEXCEK L., CERCE~: B., SHAH G. P. & LAJTHA L. G. (1974) Inhibitors of haemopoietic cell proliferation; reversibility of action. Br. J. Cancer 29, 407. LOaD B. I., TESTA N. G., HENDRV J. H. (1975) The relative spatial distributions of CFU~ and CFU¢ in the normal mouse femur. Blood 46, 65. CaOIZATH., FPJNDEL E. & TumANA M. (1970) Proliferative activity of the stem cells in the bone-marrow of mice after single and multiple irradiations (total or partial-body exposure). Int. J. Radiat. Biol. 18, 347. GXDALXJ. & L/OTHA L. G. (1972) Regulation of haemopoietic stem cell turnover in partially irradiated mice. Cell Tiss. Kinet. 5, 147-157. RENClUCCAN. J., Rxzzou V., HOWARD D., DUFFV P. & STOHLMANF., Jr. (1970) Stem cell migration and proliferation during severe anaemia. Blood 36, 764. LORDB. I., MORt K. J., WmC3HTE. G. & L~JI"SA L. G. (1976) An inhibitor of stem cell proliferation in normal bone marrow Br. J. Haem. 34, 441. LAJTHAL. G. (1975) Aetiological factors in marrow damage, in leukaemia and myelofibrosis. Advances in Biosciences 16, 145.
DISCUSSION HARXnSS: I have a question to Dr. Hagenbeek. You suggested that the CFU-s go from the bone marrow into the spleen because they find a better environment there. But the spleen is surely also heavily involved with the leukaemia. Have you investigated the CFU-s content at a stage when the spleen is full of leukaemic cells? HAGENBEEK : T h e phenomenon you are referring to has been observed at day 23 after inoculation. In fact, this is the preterminal stage, where the spleen consists largely of leukaemic cells. HARRXSS: Why then would the spleen be a better site than the bone marrow?
HAGENBEEK: We think that the bone marrow which is the tissue to be replaced by ieukaemic cells, loses its optimal environment for normal haemopoiesis. At that time, the spleen and the liver, which are not so heavily infiltrated, might offer a relatively more suitable condition for stem cells to lodge and to continue to proliferate. An alternative explanation might be that dormant primitive haemopoietic cells present in the liver and in the spleen, would start to proliferate upon a certain stimulus. This would imply the existence of a feedback mechanism between the bone marrow and potential extramedullary sites of blood cell production. FLIEDNER: I have one question with respect to the curve about the myeloblasts proliferation and promyelocytes proliferation. If I took it from your curve, there is a
156
L . G . L~rntA, B. I. LOitD, J. M o ~ and E. WRIGHT
difference in the rising part of about 8 days or so. Do you really think that this is representing transition from one population to the other, is it not more likely that there is an actual cell replication at various levels? I have some difficulties to understand what happens here; 8 days is simply too long to allow for this and other factors must be involved. HAGENBEEK: I think we are dealing with both proliferation and differentiation. Apparently the transition time which is needed for a leukaemic blast cell (LBC) to differentiate into a promyelocyte is much less than 8 days. Our present working hypothesis is that during the early stage of the disease only a small fraction of newly produced LBC differentiates. As the bone marrow becomes completely filled up with LBC, this fraction increases, so that from day 20 onwards the majority of the leukaemic cell population consists of promyelocytes. The distinct cell kinetic parameters of both populations remain to he investigated. KILLM^NN: It is just a continuation of Dr. Fliedner's question. As no doubt you know very well, there are highly differentiated acute myeloid leukaemias that along their way do dedifferentiate and become blastic. However, I have never seen anything go the other way round and become more differentiated as it grows. HAGENBEEK : If in a patient at the time of diagnosis the leukaemia is classified, e.g. as a promyelocytic leukaemia it might well be that at the preclinical stage the leukaemia should have been of the blastic type. However, this initial blastic phase is not detected in the patient as it is in our B N M L rat leukaemia model. LAJTHA: In Oxford some time ago we had three cases of erythro-leukaemia which presented first as a pure myeloblastic picture and were acknowledged as myeloblastic leukaemia, but all of them eventually died with a classic di Guglielmo disease. Pougs£^u: I should like to ask Dr. l-lagenbeek a question concerning the double cell population in BNM L leukaemia. I have tried to grow these cells in culture and find certain growth requirements: they proliferate in vitro in the presence of mercaptoethanol and cysteine. Both stimulators seem to be necessary. This type of cell
survives for 6 days in culture. Do you think that it is promyelocytic ? (shows microphotograph of cells). HAGENBEEK: It might b~. FL~EDNT.X: These are not promyelocytes. You cannot call them by any means promyelocytes in the rat. This is an abnormal cell in the rat, but not a promyelocyte--it is a leukaernia cell. Poup,xE^v: After inoculation into BN rats, these cultures produce leukaemia leading to death on day 28. HAGENBEEK: HOW did you exactly culture the cells, what were the requirements ? PDURgEAU : Enriched NCTC 135 M E D I U M with added vitamins and glutamine which is also our basic medium for in vitro growth of L5222 ceUs. 2-Mercaptoethanol. (5 x 10-6 M) and L-cysteine ( 2 x 10- a M) are necessary to maintain B N M L cells for a week. I have not maintained them for longer than 6 days. However, I do observe mitoses, sometimes up to 5 or 6 ~o of the cell population. MULLER-BIL,~,XT: We have to come back to the story of the migration of normal stem cells from the bone marrow to the spleen. It is important you have shown this because there is one type of acute human leukaemia where we know from agar culture measurements that there is a migration of normal CFU-c at least into the peripheral blood, namely acute lymphoblastic leukaernia. 1.6WENBERG: We have studied 7 acute myeloid leukaemic patients with the Robinson culture assay. In addition, we have used a colony forming assay for lymphocytic cells (PHA-stimulated cultures). In all these 7 patients myeloid colony forming cells were almost completely absent while there was no reduction of lymphocytic colony forming cells. This indicates in fact that the disappearance of normal myelopoiesis is not just a matter of physical expulsion of normal haemopoietic cells from the marrow due to lack of space by an overgrowing tumour mass, but to a more specific process of growth inhibition. A second remark: we have tested in two experiments the B N M L leukaemia in these 2 assays. As compared to the human data we have found a clear discrepancy. In the rat system, the disappearance of myeloid colony forming cells simply correlated with that of lymphocytic colony forming cells.
GROWTH REGULATIONS IN N O R M A L A N D LEUKAEMIC HAEMOPOIETIC STEM CELLS L. G. LAJTHA, B. I. LORD,J. MoRI and E. WRIGHT Paterson Laboratory, Christie Hospital and Holt Radium Institute, Manchester, U.K. THE QUESTIONraised by this conference is whether leukaemic cells have any 'controlling' effects on normal stem cells (and on the normal baemopoiesis in general) and whether leukaemic cells obey----even in part--the normal regulatory mechanism of the haemopoietic system. The fact that in leukaemia not infrequently normal haemopoietic elements decrease in number has been known and in animal leukaemia it has been demonstrated ([1] Fig. 1). The question, of course, arises whether such effects are due to a propensity of the leukaemic cells to produce some form of normal control or whether these effects are due to 'toxic' products of such cells. Such toxic products, theoretically, may be specific products of the cells in which case their identification could be
of diagnostic and prognostic significance. On the other hand, they could be non-specific metabolic breakdown products in the leukaemic tissue which is known to carry a significant non growth fraction and concommitant cell death. As far as the normal controlling mechanisms are concerned we can speak at two levels of control, as far as our present understanding is concerned. The first refers to certain inhibitory control materials, particularly in the 'recognisible' maturing end cells (promyelocytes, myelocytes and early normoblasts). Such materials have been described by Ryt6maa and Kiviniemi [2] and their observations have been confirmed and extended by Lord et al. [3, 4] as illustrated in Table 1. This indicates that some low LCFU in BODY
I
FRACTION d [r~'~U -'l'c'~]
01
LCFU
in SPLEEN or MARROW
lo6
ms CU~
1.0~
""
J
._~
"~
""....
\~
IJ I
i
I
l
i
i
i
,
i
I
2
3
4
S
6
7
II
9
"
DAYS after IOs LEUK.SPLEEN CELLS i.v.
.ACUm ~. normal agar colony forming units in FIG. I. LCFU, leukaemic colony forming cells; .ACU, J marrow or spleen; Repop, repopulating capacity of marrow. 153
154
L. G. LAJ'rHA,B. I. LORD,J. Morn and E. Wgiotrr TABLE 1. SPECmcrrms OF ALL EXTRACTS Factors GCE LNE NBME
IV
CFUs
CFUc
PHA lymph
n gran
CML gran
'AML'
ALL
0 ?
0 0
0 +
+ 0
+ O
+?
0 0
+
-
LI210 P388 leukaemia leukaemia 0 0
0 0
GCE, granulocyte extract; LNE, lymph node extract; NBME IV, fraction IV from normal bone marrow (non cycling); 0, no effect; +, inhibition; n gran, normal granulocytes; CML gran, granulocytes from chronic myeloid leukaemia.
molecular weight products of these cells have a highly specific feedback control effect on the proliferation of the respective cell lines. Three points must be emphasised: firstly, that the controlling materials are non cytotoxic, their action is reversible and their action is not a shut down of proliferation but simply an elongation of the respective cell cycle times. We are therefore talking about cell line specific, cell cycle length modulation. Such modulation, of course, can have very powerful effect in any amplifying transit population such as the erythroid or granulopoietic system. Taking a more or less predetermined maturation time, the number of cell cycles during that maturation process will determine the degree of amplification. Since the maturation process is essentially a 'suicide' process whereby cells will reach a non dividing state, an extra division during that time (effected by, e.g. shortening of the cell cycle) will double the output, conversely one division less (by lengthening the cell cycle times) will halve the output. The high degree of specificity of these compounds is further indicated by our observation that neither of them have any effect on the proliferation of the stem cells or of the committed precursor cells. Very little is known about proliferation in the committed precursor cells (for the erythroid or granulopoietic lines) but recently a fair amount of information became available concerning proliferation control in the pluripotent stem cell population. That some local control is operating, has been indicated by the observation that pluripotent stem cells are not randomly distributed within the marrow cavity [5]. Shielding experiments of parts of the haemopoietic tissue have also indicated that proliferation control is of a local nature [6, 7], similar conclusions were drawn in phenylhydrazine (PHZ) treated animals by Rencricca et al. [8]. Recent experiments in our laboratory indicated that the factor responsible for such local stem cell proliferation control can be easily extracted from the cells. Mixing cells from proliferating or non proliferating haemopoietic tissues indicated that a
TABLE 2. EFFECTo F CYCLING OR NON CYCLING STEM CELLS ON STEM CELL PROLIFERATION IN MIXED CELL SUSPENSIONS
SHTdR kill PHZ PHZ PHZ PHZ
BM alone BM+irr PHZ Sp. Sp alone Sp+irr PHZ BM
~40% ~15~o ~
5 ~o
~35~o
Ratio of irradiated 'signal' cells to assay cells=2:l. The irradiation (900 rad) was performed to prevent colony formation by the 'signal' cells. TABLE 3.
STIMULATORY AND INI.flBrrORY FRACTIONS FOR STEM CELL PROLIFERATION IN NORMAL A N D REGENERATING BONE MARROW
CFUs 'kill' by SHTdR 'cycling' BM Control (no addition) N BME fr. III. N B M E f r . IV. R BME fr. III.
~ 35 ~o ~ 35 ~o -*~ 5~o ~35~
'non-cycling' BM ~ 5 ~ 5 ~ 5 -*~25~
R B M E fr. I V .
~ 35 ~o
~
fr. I. and II. NBME/RBME
~ 35 ~
,~ 5
5
NBME'~extracts from normal (non-cycling) and reRBMEJgenerating (cycling) bone marrow. ft. HI "~30-50,000 and 50--100,000 tool. wt. fractions. ft. IV j respectively. compound is released from the cells. If irradicated cells from non regenerating marrow, or spleen, are mixed with regenerating stem cells (regenerating marrow) a factor profoundly inhibits the cycling of the latter. Conversely from regenerating marrows a factor appears to be released which initiates cycling of resting stem cells (E. Wright, to be published): Table 2. With brief incubation of regenerating or normal bone marrow and Amicon filtration of their cell free supernates [9] we were able to demon-
Growth regulations in normal and leukaemic haemopoietic stem cells strate the presence of a stem cell specific inhibitor fraction and f r o m regenerating marrows a stimulating fraction; Table 3. The specificity of the inhibitor is indicated by the observation that while it can completely shut down the proliferation o f pluripotent stem cells, it will not effect the cycling of, e.g. a c o m m i t t e d granulopoietic precursor. These observations indicate a practical possibility of looking at various leukaemic cell populations. Both cell line specific cell cycle modulators and the stem cell specific inhibitor can now be isolated in reasonably clean state and their effect on leukaemic cells can be investigated. Clearly appropriate extracts can be made from the various leukaemias and the presence of such factors can be looked for. This is likely to give considerable additional information on the nature of the ieukaemic process. Finally I wish to recall the fact that in C M L the pluripotent stem cells seem to be functionally normal in most respects;
155
they seem to o b e y - a t least p a r t i a l l y - - n o r m a l proliferation control [10] (Fig. 2). It will be of particular interest to study how this property of the stem cell changes during the blastic transformation.
I
c,, Ph-
.{.~1 i~lii
Both Pit- and Ph I earn diffecentiate
r.,. Ph I i ~ p l e t e l ) ,
¢~b'olled
1
diff.
FIG. 2. Scheme of stem cell population with a Ph ~ clone in it.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
TANAKAT., TESTA N. E. G. & L~u'rI-IAL. G. (1973) Leukaemic stem cell kinetics in experimental animals unifying concepts of leukaemia. Bibhhca haemat. No. 39. RVT6MAA T. & KtV]NmMX K. (1968) Control of granulocyte production. I--Chalone and antichalone, l l - specific humoral regulators. Cell Tiss. Kinet. 1, 329. LORDB. I., C-~xcEK L., CEXCEK B., ShAH G. P., DEXTER T. M. & L~JTHA L. G. (1974) lnhibitors of haemo poietic cell proliferation?: specificity of action with the haemopoietic system. Br. J. Cancer 29, 168. LORD B. I., CEXCEK L., CERCE~: B., SHAH G. P. & LAJTHA L. G. (1974) Inhibitors of haemopoietic cell proliferation; reversibility of action. Br. J. Cancer 29, 407. LOaD B. I., TESTA N. G., HENDRV J. H. (1975) The relative spatial distributions of CFU~ and CFU¢ in the normal mouse femur. Blood 46, 65. CaOIZATH., FPJNDEL E. & TumANA M. (1970) Proliferative activity of the stem cells in the bone-marrow of mice after single and multiple irradiations (total or partial-body exposure). Int. J. Radiat. Biol. 18, 347. GXDALXJ. & L/OTHA L. G. (1972) Regulation of haemopoietic stem cell turnover in partially irradiated mice. Cell Tiss. Kinet. 5, 147-157. RENClUCCAN. J., Rxzzou V., HOWARD D., DUFFV P. & STOHLMANF., Jr. (1970) Stem cell migration and proliferation during severe anaemia. Blood 36, 764. LORDB. I., MORt K. J., WmC3HTE. G. & L~JI"SA L. G. (1976) An inhibitor of stem cell proliferation in normal bone marrow Br. J. Haem. 34, 441. LAJTHAL. G. (1975) Aetiological factors in marrow damage, in leukaemia and myelofibrosis. Advances in Biosciences 16, 145.
DISCUSSION HARXnSS: I have a question to Dr. Hagenbeek. You suggested that the CFU-s go from the bone marrow into the spleen because they find a better environment there. But the spleen is surely also heavily involved with the leukaemia. Have you investigated the CFU-s content at a stage when the spleen is full of leukaemic cells? HAGENBEEK : T h e phenomenon you are referring to has been observed at day 23 after inoculation. In fact, this is the preterminal stage, where the spleen consists largely of leukaemic cells. HARRXSS: Why then would the spleen be a better site than the bone marrow?
HAGENBEEK: We think that the bone marrow which is the tissue to be replaced by ieukaemic cells, loses its optimal environment for normal haemopoiesis. At that time, the spleen and the liver, which are not so heavily infiltrated, might offer a relatively more suitable condition for stem cells to lodge and to continue to proliferate. An alternative explanation might be that dormant primitive haemopoietic cells present in the liver and in the spleen, would start to proliferate upon a certain stimulus. This would imply the existence of a feedback mechanism between the bone marrow and potential extramedullary sites of blood cell production. FLIEDNER: I have one question with respect to the curve about the myeloblasts proliferation and promyelocytes proliferation. If I took it from your curve, there is a
156
L . G . L~rntA, B. I. LOitD, J. M o ~ and E. WRIGHT
difference in the rising part of about 8 days or so. Do you really think that this is representing transition from one population to the other, is it not more likely that there is an actual cell replication at various levels? I have some difficulties to understand what happens here; 8 days is simply too long to allow for this and other factors must be involved. HAGENBEEK: I think we are dealing with both proliferation and differentiation. Apparently the transition time which is needed for a leukaemic blast cell (LBC) to differentiate into a promyelocyte is much less than 8 days. Our present working hypothesis is that during the early stage of the disease only a small fraction of newly produced LBC differentiates. As the bone marrow becomes completely filled up with LBC, this fraction increases, so that from day 20 onwards the majority of the leukaemic cell population consists of promyelocytes. The distinct cell kinetic parameters of both populations remain to he investigated. KILLM^NN: It is just a continuation of Dr. Fliedner's question. As no doubt you know very well, there are highly differentiated acute myeloid leukaemias that along their way do dedifferentiate and become blastic. However, I have never seen anything go the other way round and become more differentiated as it grows. HAGENBEEK : If in a patient at the time of diagnosis the leukaemia is classified, e.g. as a promyelocytic leukaemia it might well be that at the preclinical stage the leukaemia should have been of the blastic type. However, this initial blastic phase is not detected in the patient as it is in our B N M L rat leukaemia model. LAJTHA: In Oxford some time ago we had three cases of erythro-leukaemia which presented first as a pure myeloblastic picture and were acknowledged as myeloblastic leukaemia, but all of them eventually died with a classic di Guglielmo disease. Pougs£^u: I should like to ask Dr. l-lagenbeek a question concerning the double cell population in BNM L leukaemia. I have tried to grow these cells in culture and find certain growth requirements: they proliferate in vitro in the presence of mercaptoethanol and cysteine. Both stimulators seem to be necessary. This type of cell
survives for 6 days in culture. Do you think that it is promyelocytic ? (shows microphotograph of cells). HAGENBEEK: It might b~. FL~EDNT.X: These are not promyelocytes. You cannot call them by any means promyelocytes in the rat. This is an abnormal cell in the rat, but not a promyelocyte--it is a leukaernia cell. Poup,xE^v: After inoculation into BN rats, these cultures produce leukaemia leading to death on day 28. HAGENBEEK: HOW did you exactly culture the cells, what were the requirements ? PDURgEAU : Enriched NCTC 135 M E D I U M with added vitamins and glutamine which is also our basic medium for in vitro growth of L5222 ceUs. 2-Mercaptoethanol. (5 x 10-6 M) and L-cysteine ( 2 x 10- a M) are necessary to maintain B N M L cells for a week. I have not maintained them for longer than 6 days. However, I do observe mitoses, sometimes up to 5 or 6 ~o of the cell population. MULLER-BIL,~,XT: We have to come back to the story of the migration of normal stem cells from the bone marrow to the spleen. It is important you have shown this because there is one type of acute human leukaemia where we know from agar culture measurements that there is a migration of normal CFU-c at least into the peripheral blood, namely acute lymphoblastic leukaernia. 1.6WENBERG: We have studied 7 acute myeloid leukaemic patients with the Robinson culture assay. In addition, we have used a colony forming assay for lymphocytic cells (PHA-stimulated cultures). In all these 7 patients myeloid colony forming cells were almost completely absent while there was no reduction of lymphocytic colony forming cells. This indicates in fact that the disappearance of normal myelopoiesis is not just a matter of physical expulsion of normal haemopoietic cells from the marrow due to lack of space by an overgrowing tumour mass, but to a more specific process of growth inhibition. A second remark: we have tested in two experiments the B N M L leukaemia in these 2 assays. As compared to the human data we have found a clear discrepancy. In the rat system, the disappearance of myeloid colony forming cells simply correlated with that of lymphocytic colony forming cells.