Autocrine and paracrine growth loops in chronic lymphocytic leukemia.
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Apoptosis↗
Biomedical subjects
K Ganeshaguru
Publications and source records attributed to K Ganeshaguru.
Interferon-alpha inducible 2'-5' oligoadenylate synthetase transcripts in lymphoid and myeloid leukemias.
Alpha-interferon (IFN) is effective in the treatment of a proportion of patients with hairy cell leukemia (HCL). B-cell chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and multiple myeloma (MM). One of the proteins induced by IFN is the enzyme 2'-5' oligoadenylate synthetase (2-5 AS). Peripheral blood or bone marrow samples treated with IFN in vitro, or from patients treated with IFN were studied for expression of the different 2-5 AS mRNA transcripts. A total of four normal individuals and 31 patients (nine HCL, five CLL, six MM, nine acute myeloid leukemia (AML) and two T-cell acute lymphoblastic leukemia (T-ALL) have been investigated. In normal peripheral blood lymphocytes, only the 1.8 kb transcript was induced with IFN in vitro. In HCL, CLL, and MM all four transcript sizes were induced by IFN in vivo and in vitro. The 1.6 and 1.8 kb forms were equally and predominantly expressed in HCL and B-CLL. On the other hand, the 1.8 kb transcript was predominantly expressed in MM and this increased expression was statistically significant. In acute leukemia, the majority of samples expressed all four transcripts equally but four of eleven samples expressed only the 1.8 kb transcript. These results suggest that the pattern of induction of specific 2-5 AS mRNA transcripts may be related to the underlying disease. Whether these different patterns of 2-5 AS induction have implications for response to IFN treatment, remains to be determined.
2',5'-Oligoadenylate Synthetase↗
Increase in 2',5'-oligoadenylate synthetase caused by deoxycoformycin in hairy cell leukaemia.
2',5'-oligoadenylate synthetase (2-5OAS) has been studied in peripheral blood mononuclear cells from nine patients with hairy cell leukaemia (HCL) receiving therapy with the adenosine deaminase inhibitor deoxycoformycin (dCF) or alpha interferon (alpha-IFN). 2-5OAS mRNA was assayed by dot-blot hybridization. Increase of 2-5OAS mRNA level was seen in six patients with HCL treated with dCF and in one patient treated with alpha-IFN who responded to therapy. A patient with a variant form of HCL treated with dCF and the second patient treated with alpha-IFN did not show an increase of 2-5OAS mRNA and neither responded to therapy. The 15 other patients with T or B-chronic lymphoblastic leukaemia (CLL), T-acute lymphoblastic leukaemia (ALL), adult T-cell leukaemia lymphoma (ATLL), non-Hodgkins lymphoma (NHL), Sezary and T or B-prolymphocytic leukaemia (PLL) treated with dCF did not show an increase in 2-5OAS, though four patients, all with T-cell tumours, responded clinically. 2-5OAS activity is known to be stimulated by alpha-IFN and recent work suggests that this rise in 2-5OAS may result in increased cleavage of mRNA for tumour necrosis factor (TNF) and other cytokines on which autocrine growth and proliferation of the tumour cells are dependent. By analogy, we suggest that one mechanism of action of dCF in hairy cell leukaemia may be to break down an autocrine growth loop for TNF or other cytokines. An alternative explanation for these observations is that cytokines released from hairy cells in the bone marrow killed by dCF induce a rise in 2-5OAS in circulating leucocytes.
2',5'-Oligoadenylate Synthetase↗
Effects of interferon-alpha (IFN) on the expression of interleukin 1-beta (IL-1), interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF) in acute myeloid leukemia (AML) blasts.
Interferon-alpha (IFN) induces the enzyme 2-5 oligoadenylate synthetase (2-5 AS) in cells from patients with hairy cell leukemia and B-cell chronic lymphocytic leukemia and this is associated with a breakdown of certain species of cytokine messenger (m)RNA via the activation of a latent ribonuclease. We have studied the expression of the cytokines interleukin 1-beta (IL-1), interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumour necrosis factor alpha (TNF) as well as of the ribonuclease activator 2-5 AS in the presence and absence of IFN in acute myeloid leukaemia (AML) blast cells from 26 patients. Before monocyte and T-cell depletion there was no expression of IL-1, IL-6 or GM-CSF, and only three of 13 patients studied expressed TNF mRNA. After cell depletion one or more cytokine was expressed in 31-62% of the 26 patients. Expression of one or more mRNA for IL-1, IL-6, GM-CSF and TNF after 18 h incubation was detected in 16 of 26 patients (63%) and this was particularly so in French-American-British (FAB) subtypes M4 and M5. Eight of nine patients with IL-6 mRNA expression and seven of 10 with IL-1 mRNA expression were in the FAB subtypes M4 and M5. Twenty-two of 26 patients showed induction of 2-5 AS mRNA in response to IFN in vitro. Exposure to IFN resulted in reduction of IL-1 mRNA in nine of 12 cases, of IL-6 mRNA in eight of nine, and GM-CSF mRNA in five of seven cases. TNF mRNA was unaffected by IFN despite 2-5 AS induction in 12 of 13 patients expressing this cytokine. In the presence of exogenous IFN, cells from six of seven patients studied showed inhibition of 3H-thymidine incorporation into DNA. DNA synthesis could also be abrogated in six of seven patients with anti-IL-1 monoclonal antibodies (MoAb) and in two of seven with anti-IL-6 MoAb. This inhibitory effect could be reversed in all patients when anti-IL-1 or anti-IL-6 was given in combination with their corresponding cytokine. These data suggest that IFN may exert a therapeutic effect in a proportion of AML patients by blocking IL-1 and IL-6 mediated growth, consequent on activation of the ribonuclease activator 2-5 AS.
2',5'-Oligoadenylate Synthetase↗
Stages of T-cell receptor protein expression in T-cell acute lymphoblastic leukemia.
In this study five monoclonal antibodies (MoAbs) to T-cell receptor (TCR) proteins (WT31, alpha F1, beta F1, TCR delta-1 and delta TCS-1) were used to identify discrete maturative stages in 40 cases of T-cell acute lymphoblastic leukemia (T-ALL). These MoAbs reacted exclusively with CD3+ T cells and did not label B-lineage and myeloid cells. In 17 of the 40 T-ALL cases studied the leukemic blasts lacked membrane and cytoplasmic TCR chains (group I). In 12 cases cells did not have membrane CD3/TCR but expressed cytoplasmic TCR proteins heterogenously: nine cases had cytoplasmic TCR beta chains (beta F1+, alpha F1-; group II), one case had cytoplasmic TCR alpha chains (alpha F1+, beta F1-; group III), and two cases were labeled by both alpha F1 and beta F1 MoAbs (group IV). The remaining 11 cases were mCD3+: nine were TCR alpha beta+ (group Va) and two exhibited TCR gamma delta (TCR delta-1+, delta TCS-1+; group Vb). The analysis of the TCR beta, -gamma, and -delta gene configurations in 23 of the 40 T-ALLs showed that: (1) the lack of TCR protein expression was due to the lack of TCR gene rearrangements only in one of nine cases; (2) five of five TCR beta+, TCR alpha- cases studied had germline TCR alpha genes (ie, no detectable TCR delta gene deletions); (3) seven of eight cases with TCR delta gene deletions expressed TCR alpha proteins, whereas in 12 of 20 of the T-ALLs with TCR beta gene rearrangements the synthesis of the corresponding protein occurred; only 2 of 16 cases with rearranged TCR delta genes expressed TCR delta chains. The T-ALL categories identified with anti-TCR MoAbs did not have additional characteristic phenotypic patterns and may correspond to the normal stages of T-cell development more precisely than those defined by other differentiation antigens.
Adult↗
Increase in 2',5'-oligoadenylate synthetase caused by deoxycoformycin in hairy cell leukaemia.
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2',5'-Oligoadenylate Synthetase↗
Possible mechanism of action of interferon alpha in chronic B-cell malignancies.
Recent evidence suggests that tumour necrosis factor alpha (TNF) is an autocrine growth factor for the chronic B-cell malignancies hairy cell leukaemia (HCL) and some cases of B-chronic lymphocytic leukaemia (B-CLL). Incubation with TNF in vitro has been shown to increase viability, DNA synthesis and the expression of the protooncogenes myc, fos and jun in the tumour cells from these patients. TNF in vitro also increases expression of TNF-mRNA, suggesting the existence of an autocrine growth loop for TNF in these cells. Current experiments are compatible with the hypothesis that interferon alpha (IFN) interferes with this autocrine growth loop in HCL and B-CLL by stimulating degradation of messenger RNAs (mRNAs) for a number of cytokines including that of TNF. This RNA degradation may be mediated through induction of the enzyme 2,5 oligo-A synthetase with consequent increased synthesis of 2,5 oligo-A which is known to stimulate the activity of a latent ribonuclease capable of degrading cytokine mRNAs. Circulating tumour-derived TNF may also contribute to the pancytopenia in HCL and B-CLL. Whether cytokine autocrine growth loops are important in other B-cell malignancies, e.g. myeloma and non-Hodgkin's lymphoma, and subject to IFN-stimulated breakdown needs further study.
B-Lymphocytes↗
Alpha interferon therapy for haematological malignancies: correlation between in vivo induction of the 2',5'oligoadenylate system and clinical response.
2',5'oligoadenylate (2-5A) synthetase mRNA and enzyme levels have been studied in peripheral blood mononuclear cells from 12 patients with chronic granulocytic leukaemia (CGL), 14 with essential thrombocythaemia (ET) and nine with chronic lymphocytic leukaemia (CLL), undergoing therapy with alpha interferon (alpha IFN). 2-5A synthetase mRNA was assayed by hybridization using the dot-blot technique and the enzyme activity was measured biochemically. A statistically significant difference was observed (P less than 0.05) between the degree of in vivo induction of mRNA by IFN alpha in the total patients between the good and intermediate responders and the poor responders. There was a similar pattern in each of the CGL, ET and CLL groups although this only reached statistical significance in the CGL group. In vitro induction of either mRNA or of enzyme activity, however, did not show a difference between the responders and poor responders in any of the patient groups. Our findings are consistent with the concept that 2-5A has an important role in the anti-tumour activity of alpha IFN and suggest that measurement of in vivo induction of 2-5A synthetase mRNA may be useful in predicting clinical response. In vitro studies, on the other hand, do not provide a reliable predictor of clinical response.
2',5'-Oligoadenylate Synthetase↗
First intron and M-bcr breakpoints are restricted to the lymphoid lineage in Philadelphia positive acute lymphoblastic leukemia.
Knowledge of the level of commitment of the target cell in hematological malignancies may have important therapeutic and prognostic implications. Cell lineage involvement was investigated in two cases presenting with acute lymphoblastic leukemia diagnosed on clinical and immunological findings and having the Philadelphia translocation t(9;22)(q34;q11). DNA from cells separated into mononuclear (lymphoid) and granulocytic fractions was hybridized with Philadelphia breakpoint-specific probes. This revealed that the breakpoint giving rise to the Philadelphia chromosome in case 1 was within the major breakpoint cluster region and in case 2 was in the first intron of the BCR gene. Rearrangement was found in the lymphoid but not the granulocyte fraction in each case. It is therefore concluded that the target cell for chromosomal change in these cases was a lymphoid committed progenitor cell, irrespective of breakpoint location.
Adult↗
Inability of poly-ADP-ribosylation inhibitors to protect peripheral blood lymphocytes from the toxic effects of ADA inhibition.
We have evaluated the effectiveness of two inhibitors of poly-ADP-ribosylation, nicotinamide and 3-aminobenzamide as rescue agents in resting and PHA-stimulated lymphocytes damaged by the combination of deoxycoformycin (dCF) plus deoxyadenosine (dAdo). Incubation with dCF (10(-5)M) and dAdo (10(-4)M) for 18 hours, inhibited protein and RNA synthesis in unstimulated lymphocytes and impaired the ability of the cells to respond to PHA stimulation or to give rise to T-cell colonies in methyl-cellulose. Predominantly dead cells using trypan blue exclusion were observed at day 4, in both unstimulated and PHA-stimulated lymphocytes, whether or not the drugs were removed at 18 hours. The number of viable cells at day 4 increased from 13.7% to 41.1% with the addition of 5 mM nicotinamide, and to 28.8% with 5 mM 3-aminobenzamide added with dCF and dAdo. Although nicotinamide was able to prevent a fall in NAD concentration for 24h (but not for 48h) and to reduce the fall of cell ATP concentration, the inhibition by dCF and dAdo of protein synthesis, RNA synthesis, ability of cells to form colonies or to respond to PHA was not reversed. We conclude that inhibition of NAD utilisation by inhibiting ADP-ribosylation with nicotinamide or 3-aminobenzamide does not protect cells in vitro from deoxyadenosine toxicity with ADA inhibition and is not likely to give significant clinical benefit in ADA deficiency.
Adenosine Deaminase Inhibitors↗
Selective toxicity of purine nucleosides to human leukaemic cells.
The in vitro cytotoxicity of various purine nucleosides and purine enzyme inhibitors, alone or in combination, and of the alkylating agent mafosfamide (Asta Z7557), incubated for 4 and 24 h have been studied in 17 leukaemic cell lines and normal bone marrow (BM). The purine nucleosides and their analogues included: 2'chlorodeoxyadenosine (CdA), 2'deoxyadenosine (AdR), 3'deoxyadenosine (3'AdR) (cordycepin), adenosine (AR), adenine arabinoside (Ara-A), deoxyguanosine (GdR) and guanine arabinoside (Ara-G). Purine enzyme inhibitors included 2-deoxycoformycin (dCF) and 8-aminoguanosine (8-AG). Cytotoxicity was based on inhibition of (i) incorporation of 3H-leucine into cell proteins and (ii) colony forming units--granulocytic/monocytic (CFU-GM) and for mixed cell colonies (CFU-GEMM). Marked and selective inhibition of T-cell growth was shown by the combinations dCF with either AdR or Ara-A, 8-AG and GdR and by CdA or Ara-G alone; these compounds even at high concentrations produced only partial inhibition of the growth of normal bone marrow CFU-GM and CFU-GEMM except for CdA which completely inhibited the formation of CFU-GEMM colonies. The combination dCF + cordycepin and alkylating agent mafosfamide were, however, toxic to all the cell lines at the concentrations employed, as well as to CFU-GM and CFU-GEMM. The high therapeutic index of some of the purine nucleosides with a relatively short exposure time makes them candidates for selective in vitro removal of residual neoplastic cells in autologous bone marrow transplantation (ABMT) for T-ALL.
Adenosine Deaminase Inhibitors↗
Enzyme activities of leukemic cells and biochemical changes induced by deoxycoformycin in vitro--lack of correlation with clinical response.
Deoxycoformycin (DCF) is a specific inhibitor of adenosine deaminase (ADA) and has been shown to be active in lymphoid neoplasms. Cytotoxicity is thought to be mediated by the accumulation of deoxyadenosine (AdR) and deoxyadenosine triphosphate (dATP) which inhibits ribonucleotide reductase and DNA synthesis in rapidly proliferating cells. Others suggested mechanisms leading to cell death particularly in non-dividing cells include depletion of ATP and NAD pools, inhibition of S-adenosylhomocysteine (SAH) hydrolase and induction of DNA strand breaks. In patients with high leukemic counts who were subsequently treated with DCF, we have studied (a) the levels of ADA, ecto-5'-nucleotidase (5NT), deoxyadenosine kinase (AdR-kinase) and SAH-hydrolase in the leukemic cells; [b) the in-vitro effects of DCF on dATP, ATP, NAD, SAH-hydrolase levels and on DNA strand breaks; and (c) the correlation between these parameters with clinical response to DCF. No significant difference in ADA, 5NT, AdR-kinase and SAH-hydrolase activities could be found between responders and non-responders. Incubation of the leukemic cells in vitro with DCF caused an inhibition of ADA, an accumulation of dATP, a moderate reduction in ATP and NAD levels, a suppression of SAH-hydrolase activity and an increase in DNA strand breaks in practically all the leukemic samples, irrespective of clinical response. Our results show that neither measurement of these enzymes nor studies of these biochemical sequelae of ADA inhibition in vitro predicts clinical responsiveness to DCF therapy.
Adenosine Deaminase Inhibitors↗
Circulating monoclonal B lymphocytes in multiple myeloma.
Peripheral blood mononuclear cells from 28 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of unknown significance (MGUS) were studied by immunoglobulin gene analysis. Clonal immunoglobulin gene rearrangements in peripheral blood mononuclear cells (PBIGRA) were demonstrated in 10 of the 28 MM patients (36%). Bone marrow and peripheral blood mononuclear cells were studied simultaneously in five of these 10 patients, and identical gene rearrangements were demonstrated in both. The incidence of such gene arrangements was higher in patients with active disease (cases at presentation or relapsed = 10/19 [47%]) compared to remission status (0/9) and higher in untreated (47%) compared to treated patients (11%) (P less than 0.05). Patients with this phenomenon had higher serum calcium levels (P less than 0.001), and higher bone marrow plasma cell counts (P less than 0.05). Serum creatinine and beta 2-microglobulin were also higher but did not reach statistical significance. None of the patients with monoclonal gammopathy of uncertain significance had gene arrangements. Our findings confirm that circulating B lymphocytes are part of the malignant clone in MM and their presence correlates with high tumour volume.
Antibodies, Monoclonal↗
Enzymes of purine metabolism in lymphoid neoplasms, clinical relevance for treatment with enzyme inhibitors.
A few enzymes of the purine degradative pathway have proved valuable in diagnosis and treatment of lymphomas and lymphocytic leukemia. Of particular interest are the enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and ecto-5'-nucleotidase (5NT). Intact activities of ADA and PNP have been shown to be vital for lymphoid cells. During development, lymphoid precursors go through remarkable changes in the concentrations of these enzymes and the neoplasms derived from them show a "frozen" biochemical profile similar to the corresponding normal cell of origin. Knowledge of the role of these enzymes has led to the pharmacological use of enzyme inhibitors for the specific treatment of lymphoid neoplasms. This review concerns the enzymatic make-up of normal and neoplastic lymphocytes and exploitation of this knowledge for the treatment of lymphomas. Special emphasis will be put on the clinical use of an ADA-inhibitor, deoxycoformycin.
5'-Nucleotidase↗
Absence of Kirsten-ras oncogene activation in B-cell chronic lymphocytic leukemia.
By using a combination oligonucleotide probe hybridization and restriction enzyme polymorphism analysis, a series of 48 cases of B-cell chronic lymphocytic leukemia were investigated for activating point mutations at codons 12, 13 and 61 of the K-ras proto-oncogene. A small series of acute leukemias (seven with acute lymphoblastic leukemia (ALL), 11 with acute myeloid leukemia (AML)) were examined in parallel. None of the cases of B-CLL contained detectable activating mutations of the K-ras gene at codon 12 (GGT-gly----GCT-ala) was detected at presentation. In both cases of acute leukemia, the mutation was restricted to one allele and could not be detected in remission samples. Those data suggest that activation of members of the ras oncogene family, typified by K-ras, may be less important in disease pathogenesis in leukemias such as B-CLL that arise from a more committed progenitor.
Aged↗
Clinical response to deoxycoformycin in chronic lymphoid neoplasms and biochemical changes in circulating malignant cells in vivo.
Deoxycoformycin (DCF), an adenosine deaminase (ADA) inhibitor, has been shown to be active in lymphoid neoplasms. The mechanism of cytotoxicity might involve accumulation of deoxyadenosine triphosphate (dATP), depletion of the nicotinamide adenine dinucleotide (NAD) and ATP pool, induction of double-stranded DNA strand breaks, or inhibition of S-adenosyl homocysteine hydrolase (SAH-hydrolase). We have investigated the biochemical changes in the circulating malignant cells of patients with chronic leukemia/lymphoma who were treated with DCF (4 mg/m2 weekly). Blood samples were taken from 17 patients with 60% or more circulating leukemic cells before, 4, 24, and 48 hours and five days after the first administration of DCF. Leukemic cells were separated and studied for changes in ADA, dATP, ATP, NAD, and SAH-hydrolase levels and DNA strand breaks and the data analyzed according to clinical response. Inhibition of ADA activity was found in all except one patient at 4 to 24 hours after the first administration of DCF. dATP started to accumulate at four hours, reached a maximum level between 24 and 48 hours, and returned to base values on the fifth day. Intracellular ATP and NAD levels were transiently reduced in some of the patients. However, no correlation between these changes and a clinical response could be found. DNA strand breaks could be studied in 13 patients. A significant increase in DNA breaks at 24 to 48 hours was found in six of the seven responders but only in one of the six nonresponders. At 24 hours, SAH-hydrolase levels were reduced in all seven responders studied, but only in two of the seven nonresponders. The difference in inhibition of SAH-hydrolase was statistically significant (P = .0023). These results suggest that DNA strand breaks and inhibition of SAH-hydrolase correlate with clinical response.
Adenosine Deaminase Inhibitors↗
Intertribal variation in the prevalence of alpha-thalassemia in Saudia Arabia.
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Epidemiologic Methods↗
Membrane phenotype and response to deoxycoformycin in mature T cell malignancies.
The adenosine deaminase inhibitor deoxycoformycin was used in low doses to treat 19 patients with clinically aggressive T cell malignancy with a mature membrane phenotype. The patients comprised eight with prolymphocytic leukaemia, two with chronic lymphocytic leukaemia, four with adult T cell leukaemia-lymphoma, three with Sézary syndrome, and two with T cell lymphoma. Two thirds of the patients had been resistant or minimally responsive to combination chemotherapy. Complete remission was obtained in five patients (two with prolymphocytic leukaemia and one each with chronic lymphocytic leukaemia, adult T cell leukaemia-lymphoma, and Sézary syndrome) and partial remission in two others. Unmaintained complete remission lasting more than one year was seen in three patients. Responses were obtained only in patients with CD4+,CD8-membrane markers (seven out of 10), and no responses were recorded in any of the nine patients with a different phenotype. In this series remission appeared to correlate with the membrane phenotype of the neoplastic cell and not with the cytopathological diagnosis. Future studies should establish the biochemical basis for the greater sensitivity of CD4+ lymphoid cells to deoxycoformycin.
Adult↗