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We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo diagnosed acute myeloid leukaemia (AML) and the relationship between presence of P-gp in leukaemic cells and efficacy, as remission induction and survival rate, of two different anthracyclines, daunorubicin (DNR) and idarubicin (IDR). We found that 30 out of 50 patients (60%) were negative (Group 1) and 20 (40%) were positive (Group 2) for P-gp expression evaluated by mean of MRK16 MoAb using a cut-off of 10% positive cells. Thirty-five out of 50 patients (70%) obtained complete remission (CR); depending on P-gp expression, the CR rate was 80% for group 1 and 45% for group 2 (p < 0.005). The median duration of overall survival was 20 months for patients in Group 1 as compared with 10 months for patients of Group 2 (p < 0.005). Regarding the anthracycline used, no significant difference in CR was observed in patients of Group 1 (75% of CR with DNR vs. 90% with IDR); Group 2 obtained 40% of CR with DNR vs. 70% with IDR (p < 0.005). The median duration of overall survival (OS) with the two regimens was comparable in Group 1, while it was significantly longer in patients of Group 2 treated with IDR compared with DNR regimen (p < 0.005). These results confirm the prognostic value of P-gp expression in AML at first appearance and we suggest that idarubicin could be a valid anthracycline drug in the treatment of AML to be evaluated as potential drug of choice in patients with primary or drug-induced multidrug resistance.

BACKGROUND Chronic myeloid leukemia is caused by a chromosomal translocation that results in an oncogenic fusion protein, Bcr-Abl. Bcr-Abl is a tyrosine kinase whose activity is inhibited by the antineoplastic drug STI571. This drug can cure mice given an injection of human leukemic cells, but treatment ultimately fails in animals that have large tumors when treatment is initiated. We created a mouse model to explore the mechanism of resistance in vivo. METHODS Nude mice were injected with KU812 Bcr-Abl(+) human leukemic cells. After 1 day (no evident tumors), 8 days, or 15 days (tumors >1 g), mice were treated with STI571 (160 mg/kg every 8 hours). Cells recovered from relapsing animals were used for in vitro experiments. Statistical tests were two-sided. RESULTS Tumors regressed initially in all STI571-treated mice, but all mice treated 15 days after injection of tumor cells eventually relapsed. Relapsed animals did not respond to further STI571 treatment, and their Bcr-Abl kinase activity in vivo was not inhibited by STI571, despite high plasma concentrations of the drug. However, tumor cells from resistant animals were sensitive to STI571 in vitro, suggesting that a molecule in the plasma of relapsed animals may inactivate the drug. The plasma protein alpha1 acid glycoprotein (AGP) bound STI571 at physiologic concentrations in vitro and blocked the ability of STI571 to inhibit Bcr-Abl kinase activity in a dose-dependent manner. Plasma AGP concentrations were strongly associated with tumor load. Erythromycin competed with STI571 for AGP binding. When animals bearing large tumors were treated with STI571 alone or with a combination of STI571 and erythromycin, greater tumor reductions and better long-term tumor-free survival (10 of 12 versus one of 13 at day 180; P

Between 1991 and 1993 we conducted a collaborative trial in adult acute lymphoblastic leukaemia. introducing an idarubicin (IDA)-containing regimen for induction and early consolidation, and increasing consolidation intensity with an autologous bone marrow transplantation phase (ABMT, patients aged 3.5 years with an off-therapy interval >1.5 years. The response rate was 44% (7/16) with IVAP-1 and 90% (72/80) with IVAP-2 (P = 0.0001), due to regimen-related toxicities. Post-remission therapy was administered as planned to most cases but protocol violation was registered in some patients eligible to ABMT and post-graft chemotherapy. The 5-year disease-free survival (DFS) rate was 31%. Multivariate analysis indicated that DFS was improved in patients receiving a transplant (11 allogeneic. DFS 70%: 32 ABMT. 36%: 37 neither, 17%: P 25 × 10 9 /l. T-cell or mature B-cell immunophenotype. and t(9:22)/t(4:11) (all P values

An early intensive anthracycline therapy can improve therapeutic outcome in adult acute lymphoblastic leukaemia (ALL) but is usually associated with marked myelosuppressive effects and significant morbidity by infections. To reduce this risk, we employed granulocyte colony-stimulating factor (G-CSF, filgrastim 5 microg/kg/d) as an adjunct to a myelotoxic, 14-day long induction regimen with idarubicin-vincristine-L-asparaginase-prednisone (IVAP). Owing to changes in study design, patients received 'late' (n = 28) or 'early' (n = 37) G-CSF from days 15 or 4 of IVAP, respectively, until resolution of severe neutropenia. Study endpoints included time to recovery from neutropenic nadir, duration of neutropenia

Aims and background Recombinant alpha-interferon has been shown to be effective in essential thrombocythemia and in thrombocytosis associated with other myeloproliferative disorders. Patients and methods Twenty-five untreated patients were enrolled in our study from May 1989 to April 1992. Recombinant alpha interferon-2b was administered at an initial dose of 2 megaunits (MU)/m2 three times a week at escalating doses to 5 MU/m2 or the maximum tolerated dose. The mean follow-up for patients still in treatment at the time of this report was 35.9 months (range, 24-63). Results Fourteen patients (56%) had achieved a complete remission by a mean time of 152 days; 6 patients (24%) had achieved a good partial remission by a mean of 180 days. In addition to the favorable effect on platelet count, a marked improvement in clinical symptoms was observed. Treatment had to be discontinued in 9 patients (36%), 5 for toxicity (3 neurologic, 1 anemia and 1 severe hypertriglyceridemia) at a median of 155 days from the beginning of therapy (range, 30-400). Four patients refused to continue therapy because of low tolerance (flu-like syndrome) at mean of 160 days from the beginning of therapy (range, 34-301). Conclusions In our study, lower doses were administered compared with previous short-time trials. The present data show that interferon is an effective alternative to cytostatic agents in long-term treatment of patients with essential thrombocythemia, even when used at lower dosages.

Fifteen patients with a primary myelodysplastic syndrome (MDS) transformed into acute myeloblastic leukemia (AML) were treated with an intensive chemotherapy regimen containing idarubicin. A complete response (CR) was obtained in 10 patients (66.6%). In five of them this was achieved after a single course of chemotherapy. The median time to achieve a CR was 32 days (range 16-42). A partial remission (PR) was obtained in 2 patients after two induction courses of chemotherapy. One patient died during the first induction course following acute respiratory distress syndrome (ARDS) complication, whereas the chemotherapy regimen failed in 2 patients. A short interval between MDS and transformation into AML was associated with a better chance of achieving a CR. Age, karyotype, type of MDS, peripheral blood or bone marrow findings appeared to have no influence on the response to treatment. The median event free survival for patients who achieved CR was 15 months and the median actuarial survival 18 months. These preliminary results need to be confirmed in a multicentre prospective study comparing idarubicin with other anthracyclines.

This study aimed to evaluate the efficacy of amikacine and ceftazidime as an empirical antibiotic therapy for neutropenic patients affected by haematological neoplasms and to investigate the presence of prognostic features suggesting a poor outcome with this antibiotic combination at the onset of infection. This could allow the identification of subgroups of patients with a low rate of response to amikacin/ceftazidime therapy; in these patients different initial empirical therapy may be indicated. The study population comprised 166 severely neutropenic (absolute neutrophil count below 500/μl) oncohaematological patients with fever or clinical signs of infection. Multivariate analysis confirmed four negative prognostic factors: 3 or more days of hospitalization at the onset of an infectious episode, a diagnosis of acute myeloid leukaemia, a haematological disease status different from complete remission, the presence of pneumonia. Depending on how many factors are present, cases can be stratified into three groups, of significantly different prognosis: favourable (0 or 1 factor) 76% success; intermediate (2 factors) 52% success; unfavourable (3 or 4 factors) 19% success. At the onset of an infectious episode a subgroup of patients with a very low response rate to empirical amikacin/ceftazidime antibiotic therapy is identifiable, for whom a different therapy is indicated. Because of the high rate of proven or probable fungal infections in this group, the immediate administration of a systemic antifungal therapy, in addition to antibacterial agents, could be considered in these high-risk patients. Studies should be specifically addressed to evaluating a stratification of empirical antibiotic therapy according to risk factors present at the onset of infection.

From the sequencing of three genomic DNA fragments and PCR amplification products from total human DNA, we have derived the sequence of a 545-bp Sau3A fragment (68% GC), representative of a family of human DNA repeats. Since previous studies suggested its linkage with unrelated Sau3A repeats of 68 bp (54% GC) (beta-satellite sequences), this feature was further investigated by in situ hybridization experiments and by Southern blot analysis of a panel of DNAs from human-Chinese hamster somatic cell hybrids. Both DNA repeats are preferentially localized on the heterochromatic regions of acrocentric chromosomes, on the pericentromeric heterochromatin of chromosome 1, 3 and 9, and on the proximal euchromatic region of the chromosome Y q arm. On chromosome 9, both repeats are part of a 2.7-kb higher-order repeat unit. These results and the Southern blot analysis on partial digests of total DNA, suggest that the linkage between the two repetitive DNA sequences is a constant feature throughout the genome. Furthermore, Southern blot analysis of HpaII-digested and MspI-digested DNA from different human tissues and tumor cell lines indicates that the investigated heterochromatic blocks appear to be subjected to changes in their methylation pattern.

Fifty two adults (aged 15 to 66 years) with newly diagnosed acute lymphoblastic leukemia (ALL, n = 47) or lymphoid blast phase chronic myelogenous leukemia (Ly-CML, n = 5) were managed with three distinct protocols containing idarubicin at a cumulative dose of 36, 20, and 10 mg/m2, respectively, plus vincristine, L-asparaginase, and prednisolone (IVAP-1, -2, -3). IVAP-1 was highly toxic and gave a low complete remission (CR) rate (7/17, 41%). Nine patients died of complications while severely neutropenic, and one had resistant disease. In contrast, 24 of 28 patients subsequently treated with IVAP-2 achieved a CR (86%, p 0.005), the rate of both hematological and extrahematological toxicity being significantly reduced compared with IVAP-1 (p0.05). With IVAP-3, 6/7 patients aged60 years achieved CR. IVAP-2 with total idarubicin 20 mg/m2 is a very effective and well tolerated regimen for the initial treatment of adults with ALL.

We studied monosomy and deletions of chromosome 7 in 170 patients with myeloid disorders and we identified a minimal region of loss in 7q31.1 spanning between the D7S2554 and D7S2460 markers. The closest gene to our most deleted microsatellite, D7S2554, is the human I-mfa domain containing (HIC) gene, alias MyoD family inhibitor domain containing (MDFIC). We investigated the involvement of HIC in myeloid neoplasms by screening for mutations the coding regions and the intron–exon boundaries of this gene in 15 patients who presented chromosome 7 deletions in the region of HIC. No mutations were found in the coding region of this gene.

Despite the positive results achieved by Imatinib mesylate (Imatinib) in the treatment of chronic myeloid leukemia (CML), over the past several years, Imatinib does not eradicate the leukemic clone. The long-term duration of response to the drug is not known. Long-term follow-up of CML patients treated with Imatinib will ultimately define the durability of such treatment and the frequency of reemergence of progressive disease. We present the results of a 6-year follow-up of 40 CML patients either in chronic or accelerated phase who obtained a durable (> 6 months) complete cytogenetic remission (CCyR) after treatment with Imatinib in a single center. In 34 cases CCyR was obtained at an Imatinib dose of 400–600 mg/day and in 6 cases after a dose increase to 600–800 mg/day. At a median follow-up of 68 months, 6 cytogenetic relapses (15%) were observed. No progressions to more advanced phases of disease have been detected during the follow-up period. Cytogenetic relapse was predicted by either a decrease in the amount of BCR-ABL transcript of less than 2 logs after the achievement of CCyR ( p = 0.0041) or a time-to-CCyR of more than 12 months ( p This 6-year follow-up of the efficacy of Imatinib therapy in CML patients who obtained a durable CCyR indicates that the relapses rate is low over this period of observation and that the rate of relapse does not increase over time.

Imatinib dose increase up to 1200 mg daily can induce new durable complete cytogenetic remissions in relapsed Ph+ chronic myeloid leukemia patients

Summary cKit and platelet-derived growth-factor receptor (PDGFR) are receptor tyrosine kinases expressed in the testis, are involved in testosterone production, and are inhibited by imatinib. We measured hormone concentrations in 38 men receiving imatinib for chronic myeloid leukaemia at baseline and during treatment. Mean follow-up was 23·6 months (SD 7·5). We noted seven cases of gynaecomastia (18%, 95% CI 6–30%). A comparison of hormone concentrations in 21 patients before and during treatment showed that patients who developed gynaecomastia had a reduction in free testosterone concentrations of 29–53 pmol/L (95% CI 11·63–47·43), while patients who did not had a decrease of 6·36 pmol/L (-1·02 to 13·74). In most men with chronic myeloid leukaemia studied here, imatinib was associated with a reduction in the production of testicular hormones and in some, with the development of gynaecomastia.

Imatinib (Glivec) is a potent inhibitor of bcr/abl, an oncogenic fusion protein that causes chronic myelogenous leukemia (CML). alpha1 acid glycoprotein (AGP) binds to imatinib with high affinity and inhibits imatinib activity in vitro and in vivo in an animal model. A pharmacokinetics analysis of imatinib was undertaken in CML patients.Imatinib plasma concentrations were measured in 19 CML patients treated with imatinib (400 or 600 mg/day). Five patients received a concomitant short-term course of clindamycin (CLI).A positive correlation between AGP and imatinib plasma levels was observed. CLI administration decreased imatinib plasma concentrations, evaluated as area under the curve (AUC) and peak concentrations (C(max)). The effects of a bolus of CLI was studied in three patients on imatinib 23 h after the last imatinib dose. Within 5-10 min in three of three cases, CLI caused a decrease in imatinib plasma concentrations of 2.6-, 2.7-, and 4.7-fold, respectively. In vitro experiments using fresh blasts from CML patients showed that AGP, at concentrations observed in the patients, decreased imatinib intracellular concentrations up to 10 times and blocked imatinib activity. The incubation with CLI restored imatinib intracellular concentrations and biological activity.AGP exerts significant effects of the pharmacokinetics, plasma concentrations, and intracellular distribution of imatinib in CML patients; these data indicate that plasma imatinib levels represent unreliable indicators of the cellular concentrations of this molecule.

Imatinib mesylate (imatinib) inhibits Bcr/Abl, an oncogenic fusion protein. The in vitro effects of imatinib on BCR/ABL+ leukemic cells include inhibition of Bcr/Abl tyrosine phosphorylation, block of proliferation, and induction of apoptosis. The in vivo effects of imatinib were evaluated in 12 CML (chronic myeloid leukemia) patients in blast crisis or accelerated phase who were treated with imatinib. Treatment caused a decrease in spontaneous proliferation of leukemic cells in 10 of 12 evaluable patients and the development of apoptosis in 9 of 11 cases. Imatinib also caused an inhibition of Bcr/Abl autophosphorylation; however, the degree of inhibition obtained in vivo was substantially lower than that achieved in vitro with similar concentrations of imatinib. In seven patients cells could be evaluated at relapse: spontaneous proliferation was no longer inhibited and Bcr/Abl phosphorylation was comparable or superior to that present at the beginning of treatment, before imatinib administration. Plasma imatinib concentrations were not reduced. Leukemic cells obtained at relapse maintained in vitro sensitivity (Bcr/Abl autophosphorylation and proliferation inhibition) to imatinib concentration measured in vivo (3 microM or higher), although a partial resistance to the antiproliferative effects of imatinib was present at low (0.01-0.3 microM) concentrations. In four patients, addition of erythromycin to blood samples obtained at relapse restored imatinib sensitivity in terms of phosphorylation inhibition, indicating that the majority of plasma imatinib was not available to cells and probably bound to alpha1 acid glycoprotein. These data suggest that measurements of Bcr/Abl kinase activity in peripheral blood samples may represent a more reliable indicator of active concentrations than the measurement of imatinib plasma levels.

We studied deletion and monosomy of chromosome 7 in 150 patients with myeloproliferative diseases. We found 8/150 patients with monosomy 7 by cytogenetics and 4/150 with deletions of the long arm of chromosome 7 by restriction fragment length polymorphism (RFLP) analysis performed with Southern and polymerase chain reaction. To overcome limitation of RFLP analysis, we restricted loss of heterozygosity study with microsatellites to 45 patients, observing deletion 7q31.1 in 7/45 patients. In all patients with molecular alterations the deletion was observed only in myeloid cells, while the monosomy was detected in both myeloid precursor and lymphocytes. This finding suggests a CD34-totipotent stem cell origin for the monosomy and a colony forming unit – granulocyte, erytrocyte, monocyte, megakaryocytes (CFU–GEMM) stem cell origin for the deletions.

We analyzed the long-term results of a phase II trial closed in 1993, including ABMT (autologous bone marrow transplant) with post-graft chemotherapy as consolidation therapy for first remission adult ALL patients. Seventy-nine patients in remission after ‘IVAP’ (idarubicin-vincristine-L-asparaginase-prednisone) were to receive 4 consolidation cycles followed by marrow harvest (unpurged), cranial irradiation, and ABMT after BCNU 300 mg/m2, etoposide 900 mg/m2, and melphalan 110 mg/m2 (restricted to patients 15–50 years-old). The ABMT regime was intentionally of „limited“ intensity, to minimize the risks of toxic death and to resume promptly continuation therapy with alternate drug pairs for 12 weeks and low-dose maintenance with mercaptopurine and methotrexate for 6 mos. (ABMT) or 18 mos. (no ABMT). At the end of early consolidation 67 patients were alive and still in CR: 10 underwent an allogeneic BMT, 32/47 aged 50 years had chemotherapy without transplants. Long-term disease-free survival was positively affected by the following variables: age

Human chronic myelogenous leukemic cells K562, carrying the Ph+ chromosome and an amplified abl oncogene, were injected subcutaneously in nude mice and gave rise to myeloid solid tumors after 4-5 weeks. DNA from the cells of the solid tumors was analyzed by Southern blotting using v-abl and actin probes. Increased amplification of human abl oncogene, but not of the actin gene was found. This suggests a clonal selection of a cell population with more copies of the abl oncogene. This in vivo selection appears to increase the tumorigenicity of K562 cells since, in a second transplantation in nude mice, we observed a shorter period of latency before tumor appearance (3 weeks) when compared to the initial transplantation.

A case of acute renal failure, due to occlusion of renal vessels in a patient with acute promyelocytic leukemia (APL) treated with all-trans-retinoic acid (ATRA) and tranexamic acid has been described recently. We report a case of acute renal failure in an APL patient treated with ATRA alone. This case further supports the concern about thromboembolic complications associated with ATRA therapy in APL patients. The patients, a 43-year-old man, presented all the signs and symptoms of APL and was included in a treatment protocol with ATRA. After 10 days of treatment, he developed acute renal failure that was completely reversible after complete remission of APL was achieved and therapy discontinued. We conclude that ATRA is a valid therapeutic choice for patients with APL, although the procoagulant tendency is not completely corrected. Thrombotic events, however, could be avoided by using low-dose heparin.

We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo acute myeloid leukemia (AML) at the initial diagnosis in order to further define the relationship between the presence of P-gp on leukemic cells and the efficacy of two different anthracycline drugs, Daunorubicin (DNR) and Idarubicin (IRR), in terms of remission, induction and survival. We found that 30 (60%) of the 50 patients were negative for P-gp expression (group 1) and 20 patients (40%) were positive (group 2) for P-gp expression by MRK16 MoAb using a cut of 10% positive cells. Among the 50 patients, 35 (70%) obtained complete remission (CR); depending on P-gp expression the CR rate was 80% for group 1 and 45% for group 2 (p

Aims and background The study assessed the role and potential benefit of rhG-CSF in reducing the frequency, duration and severity of neutropenia following cytotoxic chemotherapy according to the E-SHAP protocol and, at the same time in improving the response rate. Methods Twenty patients with resistant/relapsed intermediate or high-grade non-Hodgkin's lymphoma were treated with the E-SHAP regimen (etoposide + methyl prednisolone + high dose cytosine arabinoside and cisplatin), and in 15 of them, we administered rhG-CSF between chemotherapeutic courses. Results The 15 patients who received G-CSF after E-SHAP were neutropenic for a short time and experienced no febrile episodes or infective complications. In contrast, in the group (5 patients) who did not receive G-CSF, the WBC nadir was lower and the number of days with a neutrophil count below 1.0 × 109/L was longer, with a greater risk of inferctious complications. Of the 15 patients, only one had a delay in chemotherapy administration, and the RDI was 95% in the 65% of patients who received G-CSF. Of 5 patients treated with chemotherapy alone, 4 had a delay and the RDI was over 95% in only one patient. We obtained a good overall response rate (70%) in the group who received G-CSF. In the historical group of 5 non-Hodgkin lymphoma patients, we observed only 1 partial response and 4 had progression of disease. Conclusions Administration of G-CSF is associated with an acceleration of neutrophil recovery, indicating its potential to reduce the risk of infection. The use of G-CSF permitted us to administer intensive chemotherapy without delay and according to standard dosage, with an improved response rate.

It is well known that L-asparaginase (L-Ase) treatment may cause thrombotic events in patients with acute lymphoblastic leukemia (ALL). The mechanism of this effect is not well understood although a reduction in plasma antithrombin III (AT III) levels is observed. In our study, a group of patients treated with L-Ase received AT III concentrates as adjuvant treatment. This adjuvant treatment reduced the levels of plasma D-dimer and thrombin-antithrombin complex, which are considered as early markers of a hypercoagulability state. These preliminary data suggest that large randomized trials will have to be conducted to improve our understanding of the role of AT III concentrates in ALL therapy.

Thrombotic thrombocytopenic purpura (TTP) is a common illness characterized by platelet thrombi within the microvascularization. In its natural course, this disease has had a mortality rate of 90%. Plasma infusion or exchange achieved a survival rate of 70% to 90%. However, 10% to 30% of patients surviving the initial TTP episode relapse at regular intervals. The treatment of recurrent forms of the disease re mains a challenge; several approaches have been shown to induce medium to long term remissions. We describe a patient with recurrent TTP whose disease remitted after administration of defibrotide.

Nature Medicine 13, 13–14 (2007); corrected after print 25 January 2007 In the version of the correspondence initially published, the corresponding author's last name was stated as Gambacorti, but should have been Gambacorti-Passerini. The error has been corrected in the HTML and PDF versions of thearticle.

The clinical usefulness of our assay can be demonstrated by two typical cases: Fig. 1 gives the course of a patient who was investigated three times during CR. While the proportion of leukaemic clones was below 20% during maintenance chemotherapy it had increased to 60% 10 months later during CR. 3 months later, the patient relapsed clinically. In contrast, another patient maintained a proportion of 5-30% of phenotypically leukaemic clones at repeated investigations over a period of nearly 3 years without relapsing clinically (Fig. 2). Our data clearly demonstrate that “complete remission” of adult AML represents a balance of leukaemic and normal hematopoiesis rather than eradication of leukaemia. They argue for the action of mechanisms which suppress the outgrowth of leukaemic progenitor cells in viva. Our in vitro culture system is applicable to all cases of AML and not restricted to certain immunological constellations as the investigation of uncultured bm cells in ALL [5] and much more sensitive than the Southern blot methodology in those cases which have a gene rearrangement as a clonal marker [4, 61. Since it is relatively easy to perform it can be a valuable tool in clinical trials of postremission therapy including alternative approaches (e.g. cytokines like interleukin-2 or autologous bone marrow transplantation) as well as for testing the effectivity of in vitro purging methods.

Various restriction enzymes digest human highly repeated homogeneous DNA to discrete fragments, some of which are present in the male and absent in the female. The male specific 2.4 kilobase HaeIII fragment corresponds to human male satellite DNA IV.

By combining somatic cell genetics, in situ hybridization and Southern hybridization, we found that the c-sis oncogene in the human myelogenous leukemia cell line ML3 is translocated from the long arm (q11----qter) of chromosome 22 to the long arm (mid-portion or q21 region) of chromosome 17. This translocation does not result in rearrangement of the c-sis oncogene.

The arrangement of repetitive and non-repetitive DNA sequences was studied in the human genome. By Ag+-Cs2SO4 density gradient centrifugations of human DNA at different fragment size reannealed to different C0t values and c-RNA hybridization experiments, we have shown the presence of two repetitive DNA fractions, called fast and slow intermediate DNA, with different pattern of sequence organization. — The fast intermediate DNA sequences (6% of the genome; CsCl density in renatured form: 1.703 g/ml) are in part clustered in fragments greater then 24,000 nucleotide pairs and in part in fragments ranging from 1,800 to 600 nucleotide pairs spaced with longer more complex sequences. — The slow intermediate DNA sequences (30% of the genome; CsCl density in renatured form: 1.707 g/ml) appear to be finely interspersed with non-repetitive sequences. At a DNA fragment size of 600 nucleotide pairs only a third of the slow intermediate DNA sequences are free of unique sequences, while the other two thirds are still organized with unique sequences. — It has also been shown that a great amount of the repetitive DNA sequence transcripts in heterogeneous nuclear RNA of HeLa cells are complementary to slow intermediate DNA sequences.

Summary. Recent work has lent strong support for the viral hypothesis of pathogenesis of human leukaemia. In laboratory animals both horizontal transmission from one individual to another by inoculation of leukaemia virus in the newborn, and vertical transmission through subsequent generation, have been shown to occur. With some animal leukaemias, and in general in human leukaemia, virus-like particles cannot be detected by the electron microscope. It is generally accepted, however, that oncogenic virus infection can be transmitted through viral nucleic acids alone. Although oncogenic DNA viruses such as the Ebstein-Barr virus might be the agent of Burkitt's lymphoma, tumour viruses which cause leukaemia in animals are usually RNA, and RNA viruses are the more likely candidates as viral agents for human leukaemia. RNA molecules with nucleotide sequences related to those of animal RNA tumour viruses have been found in human leukaemia, lymphoma, sarcoma and carcinoma cells. Such RNA molecules have the size of 70S RNA and are contained in a complex with reverse transcriptase. Nucleotide sequences, related to sequences of known RNA tumour viruses, have also been detected in human leukaemic cell DNA. Viral DNA sequences appear to be integrated into the genome mainly in the DNA fraction having an intermediate repetition frequency. They are not enriched in the satellite DNAs, which are the most highly repeated sequence of the cellular genome. Normal cell genomes may contain endogenous viral sequences which are inactive and might have been transmitted vertically through many generations. Recent work indicates that inherited endogenous tumour virus nucleotide sequences are related in evolutionary related species. Therefore, it appears likely that a possible human leukaemia virus might be related to primate leukaemia viruses.

The DNA extracted from human leukemic leucocytes has been fractionated on a methylated albumin kieselguhr (MAK) column. The different fractions obtained have been reannealed to a Cot value of 20 mol times sec/1 to study the distribution of the intermediate DNA on the MAK column. Intermediate DNA contains two components, one (CsCl density after reannealing, 1.703 g/ml) obtained by reannealing high molecular weight DNA, the other (CsCl density after reannealing, 1.707 g/ml) obtained only by reannealing sonicated low molecular weight DNA. High molecular weight intermediate DNA (1.703 component) is eluted early from the MAK column in the fractions corresponding to the main DNA peak, while low molecular weight intermediate DNA (1.707 component) is more widespread on the MAK column, but appears to be enriched in the fractions eluted later. The possibility is discussed that the latter component is interspersed in that part of the genome which is apparently more homogeneous in density in an analytical CsCl gradient, and is absent on the skewed, more heterogeneous, heavy side of the main DNA in CsCl.

The hybridization of human DNA with three non-cross-hybridizing monomers (68 bp in length) of the heterochromatic Sau3A family of DNA repeats, indicates the coexistence within a Sau3A-positive genomic block of divergent Sau3A units as well as of unrelated sequences. To gain some insight into the structure of these human heterochromatic DNA regions, three previously cloned Sau3A-positive genomic fragments (with a total length of approximately 1900 base-pairs (bp)) were sequenced. The analysis of the sequences showed the presence of clustered Sau3A units with different degrees of divergence and of two DNA regions of approximately 100 bp and 291 bp in length, unrelated to the family of repeats. A consensus sequence derived from the 24 identified Sau3A monomers presents, among highly variable regions, two less variant regions of 8 bp and 10 bp in length, respectively. The Sau3A-unrelated DNA fragment 291 bp in length, used as a probe on genomic DNA digested with a series of restriction enzymes, defines a “new” family of DNA repeats possessing periodicities for HaeIII (HaeIII family). Sau3A and HaeIII repeats display a high degree of linkage in a collection of Sau3A-positive genomic recombinant phages.

Human DNA has been fractionated by centrifugation in an Ag + -Cs 2 SO 4 preparative density gradient. Besides satellite DNA I and II, previously demonstrated and characterized, a newly identified satellite DNA III has been isolated, having a CsCl density of 1.696 g/ml and accounting for 1.5 % of the total genome. The renaturation properties of human satellite DNA III, estimated by determining its CsCl densities and melting curves after denaturation and renaturation, indicate that it is fast renaturing and therefore highly repeated, as are the other human satellite DNAs. The nuclei obtained from human placenta and leukemic leucocytes have been fractionated into heterochromatin and euchromatin. Satellite DNAs are enriched in heterochromatin, while they are no longer detectable in the DNA extracted from euchromatin, centrifuged in Ag + -Cs 2 SO 4 .

The DNA extracted in macromolecular form from human leukemic leucocytes has been fractionated in Ag+- Cs2SO4 and Hg++-Cs2SO4 preparati

as well as those of mt-DNAs of many species so far examined [4-61. It has been shown that human mt- DNA molecules have a circular shape and a length of 5 rnp as in other higher organisms like other mammals, birds, amphibians, while in lower organisms, like yeasts, Neurospora, Tetrahymena and in various plants, m&DNA molecules appear to be mainly linear and of a greater length, up to 20 mlr or more [7] as reviewed recently [5]. Studies on the futition of mt- DNA were carried out by investigating protein syn- thesis in isolated mitochondria and by isolating mito- chondrial RNA species and hybridizing them with mt-DNA [g-14]. In the present investigation the genetic complexity of human mt-DNA has been evaluated by determining its renaturation kinetics. 2. Methods The mitochondria were isolated from human pla- centa, and from human leukemic leucocytes, and the mt-DNA then extracted as previously described [3] . The purity of mt-DNA was checked by centrifuging it at equilibrium in neutral CEl where it has a charac- teristic density, and in alkaline CsCl where the com- plementary strands display different densities [3]

Human DNA has been fractionated in Ag + Cs 2 SO 4 and Hg 2+ Cs 2 SO 4 preparative density gradients, and the fractions obtained have been centrifuged in neutral CsCl after extensive dialysis to eliminate Hg 2+ and Ag 2+ . By centrifugation in Ag + Cs 2 SO 4 a new satellite, called satellite DNA II, has been isolated from human DNA. It has a density of 1.693 g/ml. in neutral CsCl, accounts for 2% of the total approximately, renatures rapidly and separates into complementary strands having different densities in alkaline CsCl. In Hg 2+ Cs 2 SO 4 gradients human DNA appears to be composed of two classes of molecules. The first, which accounts for approximately 80% of the total, is highly heterogeneous in base composition, its density in CsCl ranging from 1.690 to 1.720 g/ml., and is distributed in Hg 2+ Cs 2 SO 4 so that the A·T-rich fractions are on the heavy side and the G·C-rich fractions on the light side, as expected on the basis of the preferential binding of Hg 2+ to A·T pairs. The second class, which accounts for approximately 15% of the total, is more homogeneous, has a density of 1.696 g/ml., and is located on the light side of the DNA band in the Hg 2+ Cs 2 SO 4 gradient. This suggests that the amount of Hg 2+ bound to this A·T-rich DNA is abnormally low. This second class of DNA has been isolated by preparative CsCl centrifugation from a pool of the light fractions obtained from DNA-Hg 2+ Cs 2 SO 4 centrifugation. It tends to renature after heat-denaturation, as shown by the shift of its density towards the native value in neutral CsCl.

Some clinical aspects in 18 patients affected by aplastic anemia are considered. Liver involvement might be due to serum hepatitis, drug toxicity and possibly to the same etiologic factors causing apl

In situ hybridisation of radioactive complementary RNA has been used to localise the chromosomal distribution of human satellite DNA III. This DNA is found to be concentrated in paracentromeric heterochromatin mainly on chromosome 9 and in minor concentrations on chromosomes chiefly of the D and G groups.

Experiments of hybridization between mouse leukemia virus synthetic 3H-DNA probe and mouse main band and satellite DNAs indicate that there is not a higher concentration of viral sequences in the satellite DNA. On the contrary, viral sequences appear to be enriched in the fast renaturing intermediate main band DNA.

The human genome contains repetitive nucleotide sequences that are in part organized in blocks in the heterochromatin and partly interspersed among single copy genes. It is known that chromosome deletions and rearrangements occur frequently in leukemias and cancers at cytogenetic analysis. Methods of genome investigation such as analysis of DNA by restriction enzyme digestion and Southern blot hybridization could demonstrate finer genome rearrangements. Human total DNA centrifuged to equilibrium in Ag+-Cs2SO4 shows four satellite DNAs and a shoulder on the light side. From this shoulder a DNA fraction, by us previously called homogeneous main band DNA1, made up mainly by highly repeated DNA2 other than satellite DNAs, can be separated by a subsequent CsCl centrifugation.

Les donnees epidemiologiques et virologiques dont on dispose maintenant, bien qu'elles ne prouvent pas que le virus de la leucemie bovine puisse provoquer les leucemies lymphoides B humaines, montrent cependant qu'il ne peut s'agir seulement de coincidence

The inhibition of restriction enzyme digestion of lambda phage DNA by anthracyclines (i.e., adriamycin, daunomycin, epirubicin, idarubicin and esorubicin) commonly used in the treatment of human leukemia and cancer has been studied in vitro. The anthracyclines used inhibit DNA digestion by SmaI, AvaII, HaeIII, HhaI and HpaII, which cut DNA at guanine-cytosine (G-C) sequence sites, and by EcoRI, which cut DNA at adenine adenine-thymine thymine (AATT) sequence sites. Adriamycin completely inhibits DNA restriction by the indicated enzymes, daunomycin, epirubicin and idarubicin determine only a partial inhibition, while esorubicin does not inhibit DNA restriction at all. An attempt is made to correlate the extent of the in vitro interaction between anthracyclines and DNA to in vivo cardiotoxicity.

Moloney murine leukemia virus c-DNA hybridizes mainly with cellular middle repeated sequences of NIH-Swiss mouse spleen DNA, fragmented to different lengths, denatured and renatured to an intermediate value of Cot, and fractionated in an Ag+-Cs2SO4 preparative gradient suitable to separate unique, middle repeated and highly repeated DNA.

We have studied the hybridization profile of heterogeneous nuclear RNA from HeLa cells across DNA density gradients, and found that components in the high molecular weight fraction of heterogeneous nuclear RNA of HeLa cells hybridize to discrete density fractions on the light and heavy sides of the DNA. The conditions used for hybridization in this work allowed the detection of only those components in the RNA complementary to reiterated sequences in the DNA. These sequences in HnRNA are known to include double-stranded regions, which can be isolated readily. The double-stranded RNA shows a pattern of hybridization across a DNA density gradient which is similar to that of total HnRNA. It is concluded that the repeated sequences in HnRNA are complementary to clusters of repeated sequences in the DNA.

Male and female human placenta DNA was fractionated in an Ag+-Cs2SO4 density gradient. The different fractions along the gradient were analyzed by Hae III endonuclease digestion. Within the main band DNA on the light side a component having a Hae III digestion pattern similar to that of human satellite III DNA has been identified. This component which might be defined as a cryptic satellite accounts for at least 3% of the total human DNA and has a different position than human satellite III in Ag+-Cs2SO4.

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag+--Cs2SO4 density gradient centrifugation.--According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2X10(4) copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).--Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides.--Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides.--The organization of mouse genome analyzed by Ag+--Cs2SO4 density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.