- Email: [email protected]
SUPPORTIVE CARE AFTER BONE MARROW TRANSPLANTATION OR LEUKAEMIA INDUCTION
43 RESULTS OF HLA STUDIES
Underlinings show the haplotypes inherited by patient and additional HLA antigens found. HLA typing was done by standard two-stage microcytotoxicity technique, slightly modified, with a battery of 120 well-defined typing sera.’I
irradiation. Subsequently both patients had an enlarged liver and spleen associated with occasional or prolonged pancytopenia which prevented nurmal maintenance therapy. The boy remains in his initial remission; chemotherapy was discontinued in December, 1980. The girl’s general condition gradually became worse; persistent fever resistant to treatment, respiratory symptoms, and jaundice developed. She died in May, 1981, with candida sepis without evidence of malignancy. The HLA phenotypes of the patients’ peripheral blood lymphocytes (PBL) and bone marow cells and the genotypes of their parents are shown in the table. The HLA phenotype of PBL of both patients was appropriate to the haplotypes found in their parents. However, additional HLA antigens were found among the bone marrow cells of both patients-two antigens (HLA-B 15 and Cw3) in one and three antigens (Awl9, B27w4, and Cwl) in the other. Both patients had received unirradiated blood from random donors and from their parents, the girl from her mother and the boy from his father. The two additional antigens found in the girl were also present in one haplotype of the mother, so the engrafted clone may have been derived from the mother. Bone marrow cells and PBL of both patients were stimulated in mixed lymphocyte culture with the patient’s own cells and with several control cells (figure). The response of bone marrow cells toward the patients’ own PBL was raised on day 3 after the start of the culture and decreased on day 6, resembling the kinetics of a secondary response. This was in contrast to the control cells, which initiated a higher response on day 6. PBL were very little stimulated by bone marrow cells whereas they responded normally to the control cells, giving the higher response on day 6. Chronic GVH disease was verified by HLA typing of the patients’ PBL and bone marrow cells, where chimaerism was demonstrated. Mixed lymphocyte culture studies, revealing a secondary response of bone marrow cells toward PBL, support this conclusion and suggest that there is an engrafted clone in the bone marrow which is primed with the host antigens. Acute GVH disease as a result of transfusions has been described earlier where the chimaerism was seen in PBL and there were enough cells to kill the host. In our patients with chronic disease the chimaerism was seen not in the blood but in the bone marrow. We speculate that an allogenic clone had homed on to the bone marrow (and, perhaps, other lymphoid organs) but not enough cells were produced to allow them to be detected in the blood. We conclude that many of the problems in these two children were due to a GVH reaction caused by lymphocytes in blood and granulocyte transfusions. These reactions could have been prevented had the blood been irradiated. If criteria for the use of irradiated blood given in patients with leukaemia and other malignancies cannot be developed, the irradiation of all blood products containing living lymphocytes should be considered, 1. Turunen O,
Lundqvist C, Julin M, Transplantation 1979; 27: 304
Holmlund
G, Tiilikainen A, De la Chapelle A.
2 Dinsmore RE, Straus DJ, Pollack MS, Woodruff JM, Garrett TJ, Clarkson BD, Dupont B. Blood 1980; 55: 831.
Young CW,
lymphocyte culture. Patient’s PBL and bone marrow cells (BMC) were both used as stimulator and responder cells. Additional stimulator cells derived from a HLA nonidentical individual and from pooled lymphocytes of 30 healthy blood donors. Mixed
All stimulator cells were irradiated with 2000 r. The MLC was done in round bottom microplates where 6 x 104 responder cells and 6 x 104 stimulator cells were co-cultured in a medium of 10% human AB serum, antibiotics, and glutamine in RPMI 1640 in a total volume of 0 - 2 ml. Blastogenesis was assayed on day 3 and on day 6 by measuring the uptake. All samples were done on triplicates. Results are expressed as arithmetic means of triplicate cultures. The activity measured in cultures stimulated with irradiated responder cells is deducted from the results.
3H-thymidine
since no harmful effects have been found on the metabolism or function of red cells, granulocytes, or platelets.
especially
Children’s Hospital, University of Helsinki, and Finnish Red Cross Blood Transfusion Service, SF-00290 Helsinki 29, Finland
MARTTI A. SIIMES SAIJA KOSKIMIES
SUPPORTIVE CARE AFTER BONE MARROW TRANSPLANTATION OR LEUKAEMIA INDUCTION
SIR,-The article by Dr Watson and his colleagues (Oct. 31, 957) implies that bone marrow transplantation in remission is less costly in terms of supportive care than is remission induction for acute leukaemia. This comparison is specious. The transplant p.
have already gone through remission induction (RI); the of RI followed by transplant is more than that of RI alone. Furthermore, the transplant patients are highly selected; children do substantially better than adults, and the age ranges for the two groups are very disparate. Watson et al. did not give standard errors for their results or other statistical data which would help the interpretation of their conclusions. Furthermore, the comparison is flawed because the RI regimens used could, by their very design, be expected to add 7-14 days of neutropenia to the course. Most centres of which I am aware use, as we do, one intensive course of remission induction with an average duration of neutropenia of 24-28 days. The additional duration of neutropenia would, of course, add to the necessity of supportive care. I regretfully conclude that this report is meaningless, and I am concerned that it might be used as ammunition by those who wish
patients cost
44
routinely used in remission in those patients who have a donor and who otherwise qualify as candidates. Apart from the statistics (made suspect anyway by Watson and colleagues’ admission about their early experience and regarding their previous publications on marrow transplantation), it must be remembered that there is an obligatory mortality associated with bone marrow transplantation, even under ideal circumstances, and long-term studies have so far failed to demonstrate that, all other things being equal, transplantation in remission is advantageous over the best "standard" remission induction programmes.
explanations. The Ph’-positive stem cell line may have been selectively suppressed by chemotherapy with busulphan before the marrow was harvested; or the Ph-positive line might have been selectively injured during cryopreservation. This case-report may encourage those considering autologous bone marrow transplantation in the management of CML in transformation. It means, perhaps, that marrow cells should be harvested after initial therapy at a time when either the proportion of Ph’-positive marrow metaphases is at a minimum or when such cells can be selectively damaged by cryopreservation. It also raises the possibility of attempting to treat the patient by autografting with
Roswell Park Memorial Institute, Buffalo, New York 14263, U.S.A.
phase of the disease.
to
recommend, prematurely, that bone marrow transplantation be
DONALD J.
HIGBY
DISAPPEARANCE OF PHILADELPHIA CHROMOSOME AFTER AUTOLOGOUS BONE MARROW TRANSPLANTATION FOR TREATMENT OF CHRONIC MYELOID LEUKAEMIA IN ACUTE CRISIS
SlR,—Stem cells collected from the blood or marrow of patients in the chronic phase of chronic myeloid leukaemia (CML) can later be used to re-establish haemopoiesis after the patient in transformation
high dose chemotherapy and/or total body irradiation (TBI).Because the Philadelphia (Ph’) chromosome is present in the cryopreserved stem cells, marrow metaphases after autografting are almost always Ph’-positive. We report here an unusual case in which the patient was a myeloid mosaic, with both has been treated with
Ph’-positive
and Ph’-negative metaphases in the marrow when diagnosed, but showed only Ph’-negative marrow metaphases after autografting for transformation. A 33-year-old woman was diagnosed as having CML in December, 1979. Cytogenetic studies of marrow showed 6 cells Ph’-positive and 6 cells Ph’-negative. After initial busulphan
CML
was
cells
harvested and frozen. Splenectomy was done in December, 1980. Treatment was continued with hydroxyurea. In April, 1981, blast-cell transformation was diagnosed. The blast cells were morphologically myeloblasts and typed Ia-positive and c-ALL-negative. Cytogenetics the marrow showed three distinct populations: (a) 46, XX, Ph-negative (7 treatment marrow
were
cells); (b) 46, XX, Ph’-positive (12 cells); and (c) 47, XX, Ph’positive + C marker (6 cells). The patient was treated with TACC (6-thioguanine, cyclophosphamide, cytarabine, lomustine2) followed by TBI (800 cGy at a dose rate of 19 cGy/min, with lung shielding after 600 cGy). Autologous marrow cells were then reinfused at-adose of 3500 granulocyte progenitor cells (colony forming units in culture)/kg. Recovery of haemopoiesis was slow and incomplete. The leucocyte count reached 1 0 x 109 by day 44 and 9 - 4 x 109/1 by day 73; erythropoiesis was re-established but the platelet count remained consistently below 10’ 0 x 109/1. On day 95 a bone marrow biopsy showed normal to hypercellular fragments with numerous foci of erythropoiesis and granulopoiesis but no megakaryocytes. Between day 40 and day 95 the marrow was studied cytogeneticall on
three occasions. A total of 66 mitoses
were
all 46, XX, Ph -
Ph*-negative stem cells while he or she is still in the first chronic
Department of Haematology, Hôpital Saint-Antoine, 75571
Paris, France
N. C. GORIN A. NAJMAN J. VAN DEN AKKER M. AGLIETTA G. DUHAMEL
LEUKAEMIA ASSOCIATED WITH RAZOXANE
SiR,-In their letter (Dec. 12,
p. 1343) describing two cases of leukaemia developing after chemotherapy with razoxane (ICRF 159) for malignant disease, Dr Joshi and colleagues suggest that the leukaemia might have been a consequence of DNA damage (induced by the drug). The experimental evidence that they cite is that the multinucleate morphology produced by razoxane in cultured BHK 21S (Syrian hamster) cells is most like that obtained after treatment with X-irradiation or alkylating agents. Multinucleation, however, is a late event and could result from a variety of different primary causes apart from and not excluding DNA damage. I am not aware that anyone has proposed that it might be a reasonable criterion for acute
myelomonocytic
-.
assessing potential genetic damage. Although we earlier2suggested that razoxane might be capable of acylating macromolecules, including DNA, we were unable to demonstrate any such binding in studies with the 14Clabelled 3
drug. The
probable absence of mutagenic properties in razoxane is supported by our recent observation that continuous culture of four different human cell lines in low levels of razoxane for six months failed to induce any significant resistance, whereas prior exposure to the mutagenising effects of ultraviolet radiation led to the fairly rapid emergence of resistant populations. Alkylating agents require no such assistance. The mechanism of action of the razoxane class of agents is probably unique and remains unknown. Our short abstract attempted to draw attention to whatever analogies existed with the patterns of behaviour produced by conventional agents. This kind of comparison is, almost inevitably, superficial, particularly when condensed to 200 words, and it is unfortunate that one observation has been given so much undeserved weight. Cellular
Imperial
Pharmacology and Antitumour Chemistry Laboratory, Cancer Research Fund,
London WC2A 3PX
A. M. CREIGHTON
negative. Metaphases showing 46, XX, Ph 1 -positive or 47, XX, Ph positive +C were not identified. On day 96, the patient had a cerebral haemorrhage and died. There was no evidence of blast-cell transformation at necropsy. We think it probable that the treatment administered to this patient in transformation eliminated all three cytogenetic cell lines and that haemopoiesis was re-established from stem cells in the cryopreserved bone marrow. We do not know why the Ph i-negative population of stem cells preferentially repopulated the patient’s marrow to the exclusion of the Ph’-positive line that was also present at original diagnosis. There are at least two possible
1.
Goldman JM, Johnson SA, Catovsky D, Wareham NJ, Galton DAG. Autografting for chronic granulocytic leukemia. N Engl J Med 1981; 305: 700. 2 Gorin NC, David R, Stachowiak J, Salmon Ch, Petit JC, Parlier Y, Najman A, and Duhamel G. High dose chemotherapy and autologous bone marrow transplantation in acute leukemias, malignant lymphomas and solid tumors: a study of 23 patients.
Europ JCancer 1981, 17: 557-68
INTERFERON IN NASOPHARYNGEAL SECRETIONS
SIR,-I read with interest the report by Dr Isaacs and colleagues deficient interferon production by some children with recurrent respiratory-tract infections (Oct. 31, p. 950). The limit for detection
on
of interferon by their assay seems to be 10 IU/ml. I wonder what levels of interferon they detected in the nasopharyngeal secretions (NPS) of their patients. 1 Stephens TC, Creighton AM. Mechanism of action studies with ICRF 159. Effects on the growth and morphology of BHK 21S cells. Br J Cancer 1974; 29: 99 Biochemical studies on growth-inhibiting 2. Creighton AM, Birnie GD. bisdioxopiperazines I Effect on DNA, RNA and protein synthesis in mouseembryo fibroblasts. Int J Cancer 1970: 5: 47-54 in 3. Dawson KM. Studies on the stability and cellular distribution of dioxopiperazines cultured BHK 21S cells Biochem Pharmacol 1975; 24: 2249-53.
Underlinings show the haplotypes inherited by patient and additional HLA antigens found. HLA typing was done by standard two-stage microcytotoxicity technique, slightly modified, with a battery of 120 well-defined typing sera.’I
irradiation. Subsequently both patients had an enlarged liver and spleen associated with occasional or prolonged pancytopenia which prevented nurmal maintenance therapy. The boy remains in his initial remission; chemotherapy was discontinued in December, 1980. The girl’s general condition gradually became worse; persistent fever resistant to treatment, respiratory symptoms, and jaundice developed. She died in May, 1981, with candida sepis without evidence of malignancy. The HLA phenotypes of the patients’ peripheral blood lymphocytes (PBL) and bone marow cells and the genotypes of their parents are shown in the table. The HLA phenotype of PBL of both patients was appropriate to the haplotypes found in their parents. However, additional HLA antigens were found among the bone marrow cells of both patients-two antigens (HLA-B 15 and Cw3) in one and three antigens (Awl9, B27w4, and Cwl) in the other. Both patients had received unirradiated blood from random donors and from their parents, the girl from her mother and the boy from his father. The two additional antigens found in the girl were also present in one haplotype of the mother, so the engrafted clone may have been derived from the mother. Bone marrow cells and PBL of both patients were stimulated in mixed lymphocyte culture with the patient’s own cells and with several control cells (figure). The response of bone marrow cells toward the patients’ own PBL was raised on day 3 after the start of the culture and decreased on day 6, resembling the kinetics of a secondary response. This was in contrast to the control cells, which initiated a higher response on day 6. PBL were very little stimulated by bone marrow cells whereas they responded normally to the control cells, giving the higher response on day 6. Chronic GVH disease was verified by HLA typing of the patients’ PBL and bone marrow cells, where chimaerism was demonstrated. Mixed lymphocyte culture studies, revealing a secondary response of bone marrow cells toward PBL, support this conclusion and suggest that there is an engrafted clone in the bone marrow which is primed with the host antigens. Acute GVH disease as a result of transfusions has been described earlier where the chimaerism was seen in PBL and there were enough cells to kill the host. In our patients with chronic disease the chimaerism was seen not in the blood but in the bone marrow. We speculate that an allogenic clone had homed on to the bone marrow (and, perhaps, other lymphoid organs) but not enough cells were produced to allow them to be detected in the blood. We conclude that many of the problems in these two children were due to a GVH reaction caused by lymphocytes in blood and granulocyte transfusions. These reactions could have been prevented had the blood been irradiated. If criteria for the use of irradiated blood given in patients with leukaemia and other malignancies cannot be developed, the irradiation of all blood products containing living lymphocytes should be considered, 1. Turunen O,
Lundqvist C, Julin M, Transplantation 1979; 27: 304
Holmlund
G, Tiilikainen A, De la Chapelle A.
2 Dinsmore RE, Straus DJ, Pollack MS, Woodruff JM, Garrett TJ, Clarkson BD, Dupont B. Blood 1980; 55: 831.
Young CW,
lymphocyte culture. Patient’s PBL and bone marrow cells (BMC) were both used as stimulator and responder cells. Additional stimulator cells derived from a HLA nonidentical individual and from pooled lymphocytes of 30 healthy blood donors. Mixed
All stimulator cells were irradiated with 2000 r. The MLC was done in round bottom microplates where 6 x 104 responder cells and 6 x 104 stimulator cells were co-cultured in a medium of 10% human AB serum, antibiotics, and glutamine in RPMI 1640 in a total volume of 0 - 2 ml. Blastogenesis was assayed on day 3 and on day 6 by measuring the uptake. All samples were done on triplicates. Results are expressed as arithmetic means of triplicate cultures. The activity measured in cultures stimulated with irradiated responder cells is deducted from the results.
3H-thymidine
since no harmful effects have been found on the metabolism or function of red cells, granulocytes, or platelets.
especially
Children’s Hospital, University of Helsinki, and Finnish Red Cross Blood Transfusion Service, SF-00290 Helsinki 29, Finland
MARTTI A. SIIMES SAIJA KOSKIMIES
SUPPORTIVE CARE AFTER BONE MARROW TRANSPLANTATION OR LEUKAEMIA INDUCTION
SIR,-The article by Dr Watson and his colleagues (Oct. 31, 957) implies that bone marrow transplantation in remission is less costly in terms of supportive care than is remission induction for acute leukaemia. This comparison is specious. The transplant p.
have already gone through remission induction (RI); the of RI followed by transplant is more than that of RI alone. Furthermore, the transplant patients are highly selected; children do substantially better than adults, and the age ranges for the two groups are very disparate. Watson et al. did not give standard errors for their results or other statistical data which would help the interpretation of their conclusions. Furthermore, the comparison is flawed because the RI regimens used could, by their very design, be expected to add 7-14 days of neutropenia to the course. Most centres of which I am aware use, as we do, one intensive course of remission induction with an average duration of neutropenia of 24-28 days. The additional duration of neutropenia would, of course, add to the necessity of supportive care. I regretfully conclude that this report is meaningless, and I am concerned that it might be used as ammunition by those who wish
patients cost
44
routinely used in remission in those patients who have a donor and who otherwise qualify as candidates. Apart from the statistics (made suspect anyway by Watson and colleagues’ admission about their early experience and regarding their previous publications on marrow transplantation), it must be remembered that there is an obligatory mortality associated with bone marrow transplantation, even under ideal circumstances, and long-term studies have so far failed to demonstrate that, all other things being equal, transplantation in remission is advantageous over the best "standard" remission induction programmes.
explanations. The Ph’-positive stem cell line may have been selectively suppressed by chemotherapy with busulphan before the marrow was harvested; or the Ph-positive line might have been selectively injured during cryopreservation. This case-report may encourage those considering autologous bone marrow transplantation in the management of CML in transformation. It means, perhaps, that marrow cells should be harvested after initial therapy at a time when either the proportion of Ph’-positive marrow metaphases is at a minimum or when such cells can be selectively damaged by cryopreservation. It also raises the possibility of attempting to treat the patient by autografting with
Roswell Park Memorial Institute, Buffalo, New York 14263, U.S.A.
phase of the disease.
to
recommend, prematurely, that bone marrow transplantation be
DONALD J.
HIGBY
DISAPPEARANCE OF PHILADELPHIA CHROMOSOME AFTER AUTOLOGOUS BONE MARROW TRANSPLANTATION FOR TREATMENT OF CHRONIC MYELOID LEUKAEMIA IN ACUTE CRISIS
SlR,—Stem cells collected from the blood or marrow of patients in the chronic phase of chronic myeloid leukaemia (CML) can later be used to re-establish haemopoiesis after the patient in transformation
high dose chemotherapy and/or total body irradiation (TBI).Because the Philadelphia (Ph’) chromosome is present in the cryopreserved stem cells, marrow metaphases after autografting are almost always Ph’-positive. We report here an unusual case in which the patient was a myeloid mosaic, with both has been treated with
Ph’-positive
and Ph’-negative metaphases in the marrow when diagnosed, but showed only Ph’-negative marrow metaphases after autografting for transformation. A 33-year-old woman was diagnosed as having CML in December, 1979. Cytogenetic studies of marrow showed 6 cells Ph’-positive and 6 cells Ph’-negative. After initial busulphan
CML
was
cells
harvested and frozen. Splenectomy was done in December, 1980. Treatment was continued with hydroxyurea. In April, 1981, blast-cell transformation was diagnosed. The blast cells were morphologically myeloblasts and typed Ia-positive and c-ALL-negative. Cytogenetics the marrow showed three distinct populations: (a) 46, XX, Ph-negative (7 treatment marrow
were
cells); (b) 46, XX, Ph’-positive (12 cells); and (c) 47, XX, Ph’positive + C marker (6 cells). The patient was treated with TACC (6-thioguanine, cyclophosphamide, cytarabine, lomustine2) followed by TBI (800 cGy at a dose rate of 19 cGy/min, with lung shielding after 600 cGy). Autologous marrow cells were then reinfused at-adose of 3500 granulocyte progenitor cells (colony forming units in culture)/kg. Recovery of haemopoiesis was slow and incomplete. The leucocyte count reached 1 0 x 109 by day 44 and 9 - 4 x 109/1 by day 73; erythropoiesis was re-established but the platelet count remained consistently below 10’ 0 x 109/1. On day 95 a bone marrow biopsy showed normal to hypercellular fragments with numerous foci of erythropoiesis and granulopoiesis but no megakaryocytes. Between day 40 and day 95 the marrow was studied cytogeneticall on
three occasions. A total of 66 mitoses
were
all 46, XX, Ph -
Ph*-negative stem cells while he or she is still in the first chronic
Department of Haematology, Hôpital Saint-Antoine, 75571
Paris, France
N. C. GORIN A. NAJMAN J. VAN DEN AKKER M. AGLIETTA G. DUHAMEL
LEUKAEMIA ASSOCIATED WITH RAZOXANE
SiR,-In their letter (Dec. 12,
p. 1343) describing two cases of leukaemia developing after chemotherapy with razoxane (ICRF 159) for malignant disease, Dr Joshi and colleagues suggest that the leukaemia might have been a consequence of DNA damage (induced by the drug). The experimental evidence that they cite is that the multinucleate morphology produced by razoxane in cultured BHK 21S (Syrian hamster) cells is most like that obtained after treatment with X-irradiation or alkylating agents. Multinucleation, however, is a late event and could result from a variety of different primary causes apart from and not excluding DNA damage. I am not aware that anyone has proposed that it might be a reasonable criterion for acute
myelomonocytic
-.
assessing potential genetic damage. Although we earlier2suggested that razoxane might be capable of acylating macromolecules, including DNA, we were unable to demonstrate any such binding in studies with the 14Clabelled 3
drug. The
probable absence of mutagenic properties in razoxane is supported by our recent observation that continuous culture of four different human cell lines in low levels of razoxane for six months failed to induce any significant resistance, whereas prior exposure to the mutagenising effects of ultraviolet radiation led to the fairly rapid emergence of resistant populations. Alkylating agents require no such assistance. The mechanism of action of the razoxane class of agents is probably unique and remains unknown. Our short abstract attempted to draw attention to whatever analogies existed with the patterns of behaviour produced by conventional agents. This kind of comparison is, almost inevitably, superficial, particularly when condensed to 200 words, and it is unfortunate that one observation has been given so much undeserved weight. Cellular
Imperial
Pharmacology and Antitumour Chemistry Laboratory, Cancer Research Fund,
London WC2A 3PX
A. M. CREIGHTON
negative. Metaphases showing 46, XX, Ph 1 -positive or 47, XX, Ph positive +C were not identified. On day 96, the patient had a cerebral haemorrhage and died. There was no evidence of blast-cell transformation at necropsy. We think it probable that the treatment administered to this patient in transformation eliminated all three cytogenetic cell lines and that haemopoiesis was re-established from stem cells in the cryopreserved bone marrow. We do not know why the Ph i-negative population of stem cells preferentially repopulated the patient’s marrow to the exclusion of the Ph’-positive line that was also present at original diagnosis. There are at least two possible
1.
Goldman JM, Johnson SA, Catovsky D, Wareham NJ, Galton DAG. Autografting for chronic granulocytic leukemia. N Engl J Med 1981; 305: 700. 2 Gorin NC, David R, Stachowiak J, Salmon Ch, Petit JC, Parlier Y, Najman A, and Duhamel G. High dose chemotherapy and autologous bone marrow transplantation in acute leukemias, malignant lymphomas and solid tumors: a study of 23 patients.
Europ JCancer 1981, 17: 557-68
INTERFERON IN NASOPHARYNGEAL SECRETIONS
SIR,-I read with interest the report by Dr Isaacs and colleagues deficient interferon production by some children with recurrent respiratory-tract infections (Oct. 31, p. 950). The limit for detection
on
of interferon by their assay seems to be 10 IU/ml. I wonder what levels of interferon they detected in the nasopharyngeal secretions (NPS) of their patients. 1 Stephens TC, Creighton AM. Mechanism of action studies with ICRF 159. Effects on the growth and morphology of BHK 21S cells. Br J Cancer 1974; 29: 99 Biochemical studies on growth-inhibiting 2. Creighton AM, Birnie GD. bisdioxopiperazines I Effect on DNA, RNA and protein synthesis in mouseembryo fibroblasts. Int J Cancer 1970: 5: 47-54 in 3. Dawson KM. Studies on the stability and cellular distribution of dioxopiperazines cultured BHK 21S cells Biochem Pharmacol 1975; 24: 2249-53.