Biomedical subjects
Publications and source records attributed to A T Look.
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The retroviral oncogene v-fms encodes a glycoprotein whose transport to the plasma membrane is required for transformation. Tryptic digestion of microsomes from transformed cells yielded membrane-protected amino-terminal fragments 40 kd smaller than intact molecules. These fragments were glycosylated, and they included v-fms-coded epitopes expressed at the cell surface. Deletion of the predicted membrane-spanning peptide generated polypeptides that were completely sequestered within microsomes. The mutant glycoproteins acquired more asparagine-linked oligosaccharide chains than did wild-type molecules, lacked kinase activity in vitro, were not transported to the cell surface, and had no transforming activity. Thus, the membrane-spanning segment in the middle of the glycoprotein interrupts translocation of nascent chains into the endoplasmic reticulum, ultimately orienting the amino-terminal domain outside the cell and the carboxy-terminal kinase domain in the cytoplasm. These topological features are similar to those of several growth factor receptors, suggesting that v-fms transforms cells through modified receptor-mediated signals.
The feline c-fms proto-oncogene product is a 170 kd glycoprotein with associated tyrosine kinase activity. This glycoprotein was expressed on mature cat macrophages from peritoneal inflammatory exudates and spleen. Similarly, the receptor for the murine colony-stimulating factor, CSF-1, is restricted to cells of the mononuclear phagocytic lineage and is a 165 kd glycoprotein with an associated tyrosine kinase. Rabbit antisera to a recombinant v-fms-coded polypeptide precipitated the feline c-fms product and specifically cross-reacted with a 165 kd glycoprotein from mouse macrophages. This putative product of the murine c-fms gene exhibited an associated tyrosine kinase activity in immune complexes, specifically bound murine CSF-1, and, in the presence of the growth factor, was phosphorylated on tyrosine in membrane preparations. The murine c-fms proto-oncogene product and the CSF-1 receptor are therefore related, and possibly identical, molecules.
DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.
In early 1984, we treated 13 consecutive patients with acute lymphoblastic leukemia (ALL) using an induction regimen of rapidly rotated combinations of prednisone, vincristine, asparaginase, teniposide (VM-26), cytosine arabinoside, and high-dose methotrexate (MTX) followed by leucovorin rescue. The intent of this clinical trial, designated Total Therapy Study XI, is to test the hypothesis that greater initial leukemia cell kill will decrease opportunities for the development of drug-resistant mutants, with resultant improvement in the length of disease-free survival. Five patients experienced life-threatening gastrointestinal toxicity within three weeks of the start of treatment. One died. Three other patients had severe abdominal pain, abdominal distention, diarrhea, and weight loss, but not gastrointestinal bleeding. In the remaining five patients, toxicity was rapidly reversible, and each child was able to complete the planned course of chemotherapy. The study was then amended to switch high-dose MTX from the induction phase to the consolidation phase, allowing at least one week for mucosal recovery. Among the next 28 patients who were treated, none showed evidence of severe gastrointestinal toxicity. Patients now receive high-dose MTX alone as consolidation therapy and are tolerating it adequately. Drug timing should be examined critically when intensified multiple-agent regimens are being devised for initial treatment of ALL.
Using flow cytometric techniques, we determined the pretreatment distribution of DNA content in propidium iodide-stained leukemic blasts from 205 children with "standard-risk" acute lymphoblastic leukemia (ALL). Risk assignment was based on an initial WBC count less than 100 X 10(9)/L, no thymic mass, no meningeal leukemia, and lymphoblasts lacking sheep erythrocyte receptors or surface immunoglobulin. A single aneuploid leukemic line was detected in 74 cases (36.1%): 70 hyperdiploid and four hypodiploid. For hyperdiploid cases, the DNA index (DI, or ratio of the DNA content of leukemic v normal G0/G1 cells) ranged from 1.06 to 2.0 (median, 1.20). A secondary leukemic line with hyperdiploid cellular DNA content was identified in 21 cases with diploid primary lines. Children whose primary leukemic line showed a DI greater than or equal to 1.16 (n = 57) had significantly better responses to treatment than did those with either a diploid DI (n = 130; P = .002) or values in the range of 1.01 to 1.15 (n = 14; P = .001). The relative risk of failure for hyperdiploid cases with DI greater than or equal to 1.16, corresponding to greater than or equal to 53 chromosomes, was one-third that of the other two groups. Treatment responses of patients with both diploid and hyperdiploid lines were identical to those associated with single diploid lines, but significantly worse than those associated with single hyperdiploid lines with DI greater than or equal to 1.16 (P = .016). The most favorable prognostic variables selected by a Cox proportional hazards model were: DI greater than or equal to 1.16 (P = .001), white race (P = .022), WBC less than or equal to 25 X 10(9)/L (P = .032), age between 2 and 9 years (P = .075), and hemoglobin less than 7.0 g/dL (P = .094). DNA index greater than or equal to 1.16 retained its significant prognostic impact even after adjustment for other variables (P = .001). With the combination of DI greater than or equal to 1.16 and WBC less than or equal to 25 X 10(9)/L, one can identify a group of children with ALL who have a low probability of relapse when treated with current therapy. If they remain disease-free after longer follow-up, it may be advisable to treat them with less intensive, hence less toxic, chemotherapy than patients with higher WBC counts or lower DI values.
Factors contributing to the development of central nervous system (CNS) leukemia, and the impact of leukemic involvement of this site on subsequent remission length, were determined in 184 children with acute nonlymphoblastic leukemia who had been treated in two successive clinical trials. Preventive CNS therapy in both studies consisted of intrathecal methotrexate (12 mg/m2) given monthly during the first six months of therapy and then every three months until all treatment was stopped. Children with CNS leukemia at diagnosis or relapse were given intrathecal chemotherapy weekly for four weeks and then monthly throughout the remainder of the treatment course. Those continuing in complete remission received 2,400 rad cranial irradiation plus five doses of intrathecal methotrexate before cessation of therapy. The 38 children (20.7%) with CNS leukemia at diagnosis were more likely to have an initial leukocyte count greater than or equal to 25 X 10(9)/L (P = .01) and age less than 2 years (P = .03). The presence of CNS leukemia at diagnosis did not adversely affect the remission induction rate (P = .13) or the length of complete remissions (P = .73). CNS relapse ended initial remissions in 11 patients only and did not preclude subsequent long-term survival, as four of these children are off therapy and in second complete remission for 33+ to 78+ months. Three features at diagnosis were predictive of CNS relapse: monocytic or myelomonocytic leukemia (P = .002); age less than 2 years (P = .0001); and leukocyte count greater than or equal to 25 X 10(9)/L (P = .012). By stepwise Cox regression analysis, each factor was found to have independent predictive value. Despite the apparent effectiveness of intrathecal methotrexate as preventive CNS treatment, our findings indicate that more effective prophylaxis is needed for patients with features predisposing to CNS relapse.
We treated 24 children and adolescents with stage III or IV lymphoblastic non-Hodgkin's lymphoma, using a protocol designed for patients with poor-prognosis acute lymphoblastic leukemia (ALL). Early therapy consisted of teniposide plus cytarabine administered before and immediately after prednisone, vincristine, and asparaginase. The two-drug combination was also given intermittently with continuous 6-mercaptopurine and methotrexate during the first year of continuation chemotherapy. Periodic intrathecal methotrexate and delayed cranial irradiation were used to prevent central nervous system involvement. Anthracycline compounds, alkylating agents, high-dose methotrexate, and involved-field irradiation were not used in any phase of treatment. Twenty-two (96%) of the 23 evaluable patients achieved complete remission. With a median follow-up of 2 1/2 years, only four patients have relapsed; the remainder have been disease-free for eight months to more than five years. The projected four-year continuous complete remission rate is 73% for all patients and 79% for the 19 with mediastinal involvement at diagnosis. These results demonstrate that use of teniposide plus cytarabine with an otherwise conventional plan of ALL therapy is an effective approach to the treatment of childhood lymphoblastic lymphoma.
Serum lactic dehydrogenase (LDH) levels were measured at diagnosis in 293 children with "standard-risk" acute lymphoblastic leukemia (ALL) to determine the prognostic value of this biologic feature. Standard risk assignment was based on an initial leukocyte count of less than 100 X 10(9)/L, the absence of a mediastinal mass, the absence of meningeal involvement, and the presence of lymphoblasts lacking sheep erythrocyte receptors or surface immunoglobulin. Serum LDH levels ranged from 97 to 6,595 U/L, with a mean of 547 U/L. Higher LDH levels were associated with higher leukocyte counts, lower blast cell DNA indices, lower platelet counts, a larger spleen size, and nonwhite race. LDH levels were not related to the percentage of marrow S-phase cells, liver size, French-American-British (FAB) classification, hemoglobin levels, age, sex, or the presence of the common ALL antigen on marrow blasts. Patients with the highest LDH levels (greater than 1,000 U/L) were most likely to fail treatment, whereas those with the lowest levels (less than 300 U/L) had the lowest risk of failure (P less than .0001). The prognostic significance of serum LDH level was retained in a subset of patients that included only those with leukocyte counts less than 25 X 10(9)/L (P = .0018). When 11 presenting characteristics were subjected to multivariate analysis, serum LDH level was found to have independent prognostic strength, contributing clinically important information to that gained from leukocyte count. Early measurement of serum LDH could be useful in identifying a group of standard-risk ALL patients with a high relapse hazard.
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We studied the relation between the DNA content of neuroblastoma cells and the response to therapy in 35 infants under one year of age with a diagnosis of neuroblastoma. Using flow cytometric techniques, we found that in 27 cases the primary malignant stem line consisted of neuroblasts with hyperdiploid DNA content, ranging from 1.07 to 2.42 times the finding in normal diploid cells. All remaining cases had diploid stem lines. Diploidy was more common in infants with clinical Stage D neuroblastoma (metastases beyond regional lymph nodes) than in those with other, less advanced stages: 6 of 10 as compared with 2 of 25 (P = 0.003). Of 17 evaluable patients with unresectable hyperdiploid tumors, 15 had complete responses and two had partial responses to cyclophosphamide and doxorubicin; six others with diploid tumors did not respond (P = 0.00001). We also found that each of the four infants with Evans' Stage IV-S neuroblastoma, an unusual form of disseminated neuroblastoma with a relatively good prognosis, had hyperdiploid tumor cells of clonal origin. We conclude that in neuroblastoma of infants, hyperdiploidy of tumor cells is associated with a better response to chemotherapy than is diploidy.
Cytogenetic analysis of leukemic cells obtained at diagnosis from 122 patients with childhood acute lymphoblastic leukemia (ALL) disclosed chromosomal translocations in 36 cases. Two new nonrandom translocations were identified and found to be associated with specific immunophenotypes of the disease. The first, identified in 4 of 16 cases of T-cell ALL positive for sheep erythrocyte receptors (E+), involved the short arm (p) of chromosome 11 and the long arm (q) of chromosome 14 and was designated t(11;14) (p13;q13). The second, found in 7 of 23 cases with a pre-B-cell phenotype, involved the long arm of chromosome 1 and the short arm of chromosome 19; it was designated t(1;19) (q23;p13.3). A third abnormality involving a common breakpoint on chromosome 12 (band p 12) was also identified. These two new differentiation-specific translocations suggest a mechanism for aberrant expression of genes that influence lymphoid cell growth and development, as well as leukemogenesis.
The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.
Marrow blasts from children with B cell precursor acute lymphoblastic leukemia (ALL) were studied for differences in quantitative expression of the common ALL antigen (CALLA). Of 42 untreated patients, 35 had detectable amounts of CALLA by flow cytometric (FCM) analysis of J-5 monoclonal antibody binding. Using an FCM technique that provides correlated measurements of a given cell surface antigen, cell size, and DNA content, we detected increased CALLA expression as lymphoblasts moved from G0/G1 phase through S phase of the cell cycle. The density of the antigen (per unit of blast surface area) remained relatively constant over the same interval, indicating that the change was not due to S phase-specific enhancement of CALLA expression. Eight cases had hyperdiploid cellular DNA content and in seven of these, only cells with clonal abnormalities of DNA content expressed the CALLA marker. Mean amounts of CALLA for each patient ranged widely within the study group, from very high to marginally detectable. This variation had no discernible relation to cell size, stem-line DNA content, percentage of cells in S phase, or the presence or absence of cytoplasmic immunoglobulin. Results of a univariate proportional hazards analysis showed that both quantitative level of CALLA for S phase cells (P = 0.048) and white blood cell count (P = 0.012) had made significant contributions to treatment outcome. Patients with relative amounts of CALLA less than the median value for the entire CALLA+ group had a higher rate of failure, which was virtually identical to that for the seven HLA-DR+ patients whose blasts lacked detectable CALLA. The observed interpatient variation in quantitative expression of CALLA is consistent with recognized steps in B cell precursor differentiation and may be useful in distinguishing patients with a less favorable prognosis.
Ten pediatric patients with refractory leukemia received continuous infusion high-dose cytosine arabinoside (ara-C) according to one of two escalating dosage schedules: (1) a 500-mg/m2 rapid infusion loading dose followed by 3.5 g/m2 per day continuous infusion daily for four consecutive days, or (2) a 600-mg/m2 rapid infusion loading dose followed by 5.0 g/m2 per day continuous infusion daily for four consecutive days. Major toxicity at the lower dosage level was grade IV hematopoietic aplasia of three weeks' duration. At the higher dosage level, there was a prohibitive toxicity in multiple organ systems including transient noncardiogenic pulmonary edema, fungal infections, peritonitis, severe diarrhea, transaminase elevations, and one treatment-related death due to acute renal failure. In contrast to other methods of administration of high-dose ara-C, no CNS toxicity occurred. Oncolytic responses were seen in all patients and two achieved brief, partial remissions. Steady-state plasma ara-C concentrations were 13 to 40 mumol/L at the 3.5-g/m2 dosage level and 10 to 225 mumol/L at the 5-g/m2 dosage level; CSF concentrations at both dosages ranged from 2 to 5 mumol/L. Intracellular levels and ratios of 1-beta-D-arabinofuranosylcytidine-5' triphosphate and endogenous deoxycytidine 5' triphosphate in marrow blasts varied widely at steady state during infusion. No positive correlation existed between steady-state plasma ara-C levels, toxicity, oncolytic effect, or intracellular nucleotide concentration.
With the use of cells doubly labeled with 51Cr and 125I, an analysis was made of the distribution and lysis in normal and immune syngeneic C58 mice of weakly immunogenic malignant lymphocytes (Ib) and a highly immunogenic variant (IbN) derived from them. The results showed that 51Cr release was not a valid measure of tumor cell lysis in vivo. Prior exposure to 55 degrees C for 30 minutes caused Ib to lyse immediately after iv injection. Prior X-irradiation (10,000 R) enhanced the lysis of Ib in vivo but had only minor effects on IbN. In normal mice 125I-labeled Ib were entrapped in the lungs (approximately to 95%) immediately after iv injection, released in a diphasic manner, and then accumulated in the liver, spleen, bone marrow, and lymph nodes. In immune mice initial entrapment of Ib in the lungs was only about 42%, and label did not accumulate in the described organs. Heat-inactivated Ib were not retained to a significant degree in any of the organs of normal or immune mice. IbN were retained initially (2 hr) at values lower than Ib in the whole body, lungs, and livers of normal mice. 125I-labeled Ib were used to analyze how they were distributed and lysed in normal and immune mice during the first 6 hours after iv injection. To account for the loss of 125I from a tissue because of cell lysis (as distinct from cell redistribution), the amount of 125I associated with cells and blood plasma, and lost from the whole body, was quantified. A significant loss of 125I from the whole body began at 2 hours and continued at a linear rate thereafter. Non-cell-associated 125I occurred in the blood plasma at 1-2 hours and increased at a linear rate thereafter. These results made it possible to distinguish between loss of 125I from a tissue because of cell lysis as distinct from the redistribution of intact labeled cells.
The ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis. Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis. We describe the measurement of cellular DNA distributions of megakaryocytes by two-color FCM in unfixed, unfractionated marrow--a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days. Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution. The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded. We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.