Aberrant histone modifications induced by mutant ASXL1 in myeloid neoplasms.

An epigenetic modulator Additional sex combs-like 1 (ASXL1) is recurrently mutated in myeloid neoplasms such as myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). ASXL1 mutations are also frequently detected in clonal hematopoiesis with indeterminate potential (CHIP), which is the clonal expansion of premalignant hematopoietic cells without any evidence of hematological malignancies. Thus, understanding the roles of ASXL1 in hematopoiesis and myeloid neoplasms is a clinically crucial issue. ASXL1 mutations in hematological neoplasms are typically frameshift or nonsense mutations and occur near the 5' end of the last exon, thereby the transcripts would escape from nonsense-mediated decay, Indeed, we identified the C-terminally truncated mutant protein of ASXL1 in several cell lines derived from patients with myeloid leukemia. In mouse models, expression of the mutant ASXL1 results in impaired hematopoiesis and promotes development of myeloid neoplasms. In addition, recent findings from biochemical analysis have demonstrated that the mutant ASXL1 protein gains new functions including enhancing catalytic activity of BRCA1-associated protein 1 (BAP1), resulting in reduction of H2AK119ub and aberrant gene expression essential for myeloid transformation. In this review, we will focus on the pivotal roles of the mutant ASXL1 on histone modifications and myeloid transformation.

Chaetominine reduces MRP1-mediated drug resistance via inhibiting PI3K/Akt/Nrf2 signaling pathway in K562/Adr human leukemia cells.

Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressed MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line.

Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner.

Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.

Postinhibitory rebound neurons and networks are disrupted in retrovirus-induced spongiform neurodegeneration.

Certain retroviruses induce progressive spongiform motor neuron disease with features resembling prion diseases and amyotrophic lateral sclerosis. With the neurovirulent murine leukemia virus (MLV) FrCasE, Env protein expression within glia leads to postsynaptic vacuolation, cellular effacement, and neuronal loss in the absence of neuroinflammation. To understand the physiological changes associated with MLV-induced spongiosis, and its neuronal specificity, we employed patch-clamp recordings and voltage-sensitive dye imaging in brain slices of the mouse inferior colliculus (IC), a midbrain nucleus that undergoes extensive spongiosis. IC neurons characterized by postinhibitory rebound firing (PIR) were selectively affected in FrCasE-infected mice. Coincident with Env expression in microglia and in glia characterized by NG2 proteoglycan expression (NG2 cells), rebound neurons (RNs) lost PIR, became hyperexcitable, and were reduced in number. PIR loss and hyperexcitability were reversed by raising internal calcium buffer concentrations in RNs. PIR-initiated rhythmic circuits were disrupted, and spontaneous synchronized bursting and prolonged depolarizations were widespread. Other IC neuron cell types and circuits within the same degenerative environment were unaffected. Antagonists of NMDA and/or AMPA receptors reduced burst firing in the IC but did not affect prolonged depolarizations. Antagonists of L-type calcium channels abolished both bursts and slow depolarizations. IC infection by the nonneurovirulent isogenic virus Friend 57E (Fr57E), whose Env protein is structurally similar to FrCasE, showed no RN hyperactivity or cell loss; however, PIR latency increased. These findings suggest that spongiform neurodegeneration arises from the unique excitability of RNs, their local regulation by glia, and the disruption of this relationship by glial expression of abnormal protein.

C/EBPα is an essential collaborator in Hoxa9/Meis1-mediated leukemogenesis.

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.

Accelerated leukemogenesis by truncated CBF beta-SMMHC defective in high-affinity binding with RUNX1.

Dominant RUNX1 inhibition has been proposed as a common pathway for CBF leukemia. CBF beta-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBF beta-SMMHC fusion in AML patients lacks HABD. Here, we report that the type I CBF beta-SMMHC protein binds RUNX1 inefficiently. Knockin mice expressing CBF beta-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia-initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBF beta-SMMHC.

[Characteristics of the morphofunctional condition of cell populations by the modal cell class in response to transplantation of lymphocytic leukemia p-388].

On the model of transplanted leukemia p-388, cytophotometry has shown that tumors' impact on the body includes two stages: direct affection of the target organ, indirect affection through changes in functional relations with cell populations of other organs due to the impact of transformed cells of the damaged target organ. Moreover, the progress of tumor growth alters functional relations between the organs.

38th Annual Meeting of the International Society of Experimental Hematology.

The International Society of Experimental Hematology holds its annual meeting every northern summer. This year the meeting comprised of eight plenary sessions with distinguished invited speakers on in vivo imaging and tracking of hematopoietic stem cells (HSCs), HSC niches, epigenetic regulations of stem cells and regulation of stem cell fate, leukemogenesis, and mesenchymal stem cells. The small size of the meeting (300 attendees) permitted excellent discussion and face-to-face contacts between students, junior scientists and experts. Owing to the large number of keynote speakers, this report focuses on the most novel, unpublished data presented during the meeting.

Notch3: from subtle structural differences to functional diversity.

The Notch3 gene was identified, at the beginning of 90s, as the third mammalian Notch and was initially reported as being expressed in proliferating neuroepithelium. Since then, increasing evidence has demonstrated a number of structural and functional differences between Notch3 and both Notch1 and Notch2, which exhibit the highest structural similarity among the four mammalian Notch receptors. Possibly due to its more restricted tissue distribution, targeted deletion of murine Notch3 does not lead to embryonic lethality as is observed with targeted deletion of Notch1 and Notch2. However, genetic mutation, amplification and deregulated expression of Notch3 have been correlated with the disruption of cell differentiation in transgenic mice and to development of diseases in mice and humans. This review discusses the possible relationships between the structural differences and the nonredundant roles that Notch3 plays in the pathogenesis of the human disease cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy and in the regulation of murine T-cell differentiation and leukemogenesis.

Restoration of energy metabolism in leukemic mice treated by a siddha drug--Semecarpus anacardium Linn. nut milk extract.

Chronic myeloid leukemia (CML) is a clonal disorder characterized by proliferation of hematopoietic cells that possess the BCR-ABL fusion gene resulting in the production of a 210 kDa chimeric tyrosine kinase protein. CML, when left untreated, progresses to a blast phase during which the disease turns aggressive and shows poor response to known treatment regimens. We have studied a Siddha herbal agent, Semecarpus anacardium Linn. nut milk extract (SA) for its antileukemic activity and its effect on the changes in energy metabolism in leukemic mice. Leukemia was induced in BALB/c mice by tail vein injection of BCR-ABL(+) 12B1 murine leukemia cell line. This resulted in an aggressive leukemia, similar to CML in blast crisis, myeloid subtype, confirmed by histopathological study and RT-PCR for the p210 mRNA in the peripheral blood, spleen and liver. Leukemia-bearing mice showed a significant increase in lipid peroxides, glycolytic enzymes, a decrease in gluconeogenic enzymes and significant decrease in the activities of TCA cycle and respiratory chain enzymes as compared to control animals. SA treatment was compared with standard drug imatinib mesylate. SA administration to leukemic animals resulted in clearance of the leukemic cells from the bone marrow and internal organs on histopathological examination and this was confirmed by RT-PCR for the p210 mRNA. Treatment with SA significantly reversed the changes seen in the levels of the lipid peroxides, the glycolytic enzymes, the gluconeogenic enzymes and the mitochondrial enzymes. These effects are probably due to the flavonoids, polyphenols and other compounds present in SA which result in total regression of leukemia and correction of the alterations in energy metabolism. Study of animals treated with SA alone did not reveal any adverse effects. On the basis of the observed results, SA can be considered as a readily accessible, promising and novel antileukemic chemotherapeutic agent.

Astrocytes survive chronic infection and cytopathic effects of the ts1 mutant of the retrovirus Moloney murine leukemia virus by upregulation of antioxidant defenses.

The ts1 mutant of Moloney murine leukemia virus (MoMuLV) induces a neurodegenerative disease in mice, in which glial cells are infected by the retrovirus but neurons are not. ts1 infection of primary astrocytes, or of the immortalized astrocytic cell line C1, results in accumulation of the ts1 gPr80(env) envelope protein in the endoplasmic reticulum (ER), with ER and oxidative stress. Notably, only about half of the infected astrocytes die in these cultures, while the other half survive, continue to proliferate, and continue to produce virus. To determine how these astrocytes survive ts1 infection in culture, we established a chronically infected subline of the living cells remaining after the death of all acutely infected cells in an infected C1 cell culture (C1-ts1-S). We report here that C1-ts1-S cells proliferate more slowly, produce less virus, show reduced H2O2 levels, increase their uptake of cystine, and maintain higher levels of intracellular GSH and cysteine compared to acutely infected or uninfected C1 cells. C1-ts1-S cells also upregulate their thiol antioxidant defenses by activation of the transcription factor NF-E2-related factor 2 (Nrf2) and its target genes. Interestingly, despite maintenance of higher levels of intracellular reduced thiols, C1-ts1-S cells are more sensitive to cystine deprivation than uninfected C1 cells. We conclude that some ts1-infected astrocytes survive and adapt to virus-induced oxidative stress by successfully mobilizing their thiol redox defenses.

Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukemia.

More than 70% of patients survive childhood leukemia, but chemotherapy and radiation therapy cause irreversible impairment of spermatogenesis. Although autotransplantation of germ cells holds promise for restoring fertility, contamination by leukemic cells may induce relapse. In this study, we isolated germ cells from leukemic mice by FACS sorting. The cell population in the high forward-scatter and low side-scatter regions of dissociated testicular cells from leukemic mice were analyzed by staining for MHC class I heavy chain (H-2K/H-2D) and for CD45. Cells that did not stain positively for H-2K/H-2D and CD45 were sorted as the germ cell-enriched fraction. The sorted germ cell-enriched fractions were transplanted into the testes of recipient mice exposed to alkylating agents. Transplanted germ cells colonized, and recipient mice survived. Normal progeny were produced by intracytoplasmic injection of sperm obtained from recipient testes. When unsorted germ cells from leukemic mice were transplanted into recipient testes, all recipient mice developed leukemia. The successful birth of offspring from recipient mice without transmission of leukemia to the recipients indicates the potential of autotransplantation of germ cells sorted by FACS to treat infertility secondary to anticancer treatment for childhood leukemia.

Transfection techniques affecting Stat3 activity levels.

Transfection techniques, such as calcium-phosphate or liposome-mediated gene transfer, are commonly used for the examination of the effect of a gene upon cellular phenotype and biochemical properties. We previously demonstrated that cell to cell adhesion causes a dramatic increase in Stat3 activity. Given that the opportunities for cell to cell adhesion could be altered due to the presence of the DNA-containing complexes, we examined the effect of the calcium-phosphate transfection procedure upon Stat3 activity levels. The results revealed a dramatic increase in Stat3 phosphorylation at the critical tyr705 site and Stat3 activity following calcium-phosphate transfection. This increase was noted even in the absence of DNA and was not due to the mere presence of calcium ions. In contrast, DNA introduction through electroporation or infection with a retroviral vector did not affect Stat3 activity, while cationic lipids such as lipofectamine or Fugene6 had a less pronounced effect than calcium-phosphate transfection. These results indicate that caution is required in the interpretation of results with regard to activity of Stat3 following certain commonly used transient transfection regimens.

Large-scale identification of disease genes involved in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous group of diseases in which chromosomal aberrations, small insertions or deletions, or point mutations in certain genes have profound consequences for prognosis. However, the majority of AML patients present without currently known genetic defects. Retroviral insertion mutagenesis in mice has become a powerful tool for identifying new disease genes involved in the pathogenesis of leukemia and lymphoma. Here we have used the Graffi-1.4 strain of murine leukemia virus, which causes predominantly AML, in a screen to identify novel genes involved in the pathogenesis of this disease. We report 79 candidate disease genes in common integration sites (CISs) and 15 genes whose family members previously were found to be affected in other studies. The majority of the identified sequences (60%) were not found in lymphomas and monocytic leukemias in previous screens, suggesting a specific involvement in AML. Although most of the virus integrations occurred in or near the 5' or 3' ends of the genes, suggesting deregulation of gene expression as a consequence of virus integration, 18 CISs were located exclusively within the genes, conceivably causing gene disruption.

Induced leukemia and antineoplastic agent carmustine cause permanent changes in craniofacial growth of immature rats.

OBJECTIVES: The purpose of the present study was to investigate the possible effects of untreated terminal leukemia on craniofacial growth (Study I), and also the effects of the antineoplastic agent carmustine on craniofacial growth in both leukemic and healthy rats (Study II). MATERIAL: A total of 367 inbred Piebald variegated rats was used. METHOD: Transmission of leukemic cells was carried out intraperitoneally at 30 days of age, and without treatment (Study I), the rats reached the terminal phase within 17 +/- 1 days. Rats with induced leukemia was cured with 10 mg/kg carmustine (BCNU) given on days 6 and 13 following cell transmission (Study II), the rats remaining in remission until they were killed at 100 days of age. Final weight was recorded and 12 craniofacial dimensions and tibial length were measured with a digital sliding caliper. RESULTS: The results showed that the effect of untreated terminal rat leukemia (Study I) on craniofacial growth differed between the genders. Male rats showed clearly reduced dimensions of facial structures and also retarded general body growth, whereas females showed differences mainly in general body growth. The effect of cured leukemia (Study II) as such was minor, while BCNU had a strong and permanent reducing effect on both craniofacial and general body growth in both genders. CONCLUSION: We suggest that the results in Study I came both from a direct effect of leukemia and an indirect effect of untreated terminal leukemia through malnutrition. The alkylating agent BCNU seemed to be the main cause of permanent craniofacial and general growth retardation in Study II.

The roles of FLT3 in hematopoiesis and leukemia.

FLT3 is a receptor tyrosine kinase expressed by immature hematopoietic cells and is important for the normal development of stem cells and the immune system. The ligand for FLT3 is expressed by marrow stromal cells and other cells and synergizes with other growth factors to stimulate proliferation of stem cells, progenitor cells, dendritic cells, and natural killer cells. Mutations of FLT3 have been detected in about 30% of patients with acute myelogenous leukemia and a small number of patients with acute lymphocytic leukemia or myelodysplastic syndrome. Patients with FLT3 mutations tend to have a poor prognosis. The mutations most often involve small tandem duplications of amino acids within the juxtamembrane domain of the receptor and result in constitutive tyrosine kinase activity. Expression of a mutant FLT3 receptor in murine marrow cells results in a lethal myeloproliferative syndrome and preliminary studies suggest that mutant FLT3 cooperates with other leukemia oncogenes to confer a more aggressive phenotype. Taken together, these results suggest that FLT3 is an attractive therapeutic target for kinase inhibitors or other approaches for patients with mutations of this gene.

Effects of chronic methamphetamine exposure on heart function in uninfected and retrovirus-infected mice.

Methamphetamine (MA) increases catecholamine levels, which have detrimental effects on heart function through vasoconstriction, myocardial hypertrophy, and fibrosis. Murine retrovirus infection induces dilated cardiomyopathy (DCM). The present study investigated the cardiovascular effects of chronic MA treatment on uninfected and retrovirus-infected mice. C57BL/6 mice were studied after 12 weeks treatment. The four study groups were (group I) uninfected, MA placebo; (group II) infected, MA placebo; (group III) uninfected, MA treatment; and (group IV) infected and MA treatment. MA injections were given i.p. once a day for 5 days/week with a increasing dose from 15 mg/kg to 40 mg/kg. Left ventricular mechanics were measured in situ a using Millar conductance catheter system for pressure-volume loop analysis. Cardiac pathology was determined with histological analysis. In the uninfected mice, the load independent contractile parameters, pre-load recruitable stroke work (PRSW) and dP/dt(max) vs. Ved, significantly decreased by 32% and 35% in MA treated mice when compared to the saline injected mice. In retrovirus-infected mice, although there were no significant difference in Ees, PRSW, and dP/dt(max) vs. Ved due to MA treatment, they were increased 45%, 15% and 42% respectively when compared to saline treated mice. No further lowered heart function during murine AIDS may be due to the counteraction of the retroviral DCM and the MA induced myocardial fibrosis and hypertrophy (thickening of the ventricular walls). This is supported by increases in the End-diastolic volume (Ved, 38%) and End-systolic volume (Ves, 84%) in the retrovirus-infected saline injected mice, the decreases of 33% and 17% in the uninfected MA-treated mice, but no significant changes in the retrovirus-infected MA treated mice when compared to uninfected saline injected mice. These data suggest that MA induced myocardial cellular changes compensate for retrovirus induced DCM.

Determination of a humane endpoint in the L1210 model of murine leukemia.

The purpose of our study was to derive an alternate end-point to death or moribund appearance for the frequently used L1210 model of murine leukemia. In reviewing the published literature, we were unable to identify a suitable intermediate marker of substantive disease that predicted outcome in the BDF1 recipient of the L1210 leukemia. In an attempt to refine the use of animals in our laboratory, we developed a scoring sheet for behavioral and physical changes that followed intravenous injection of L1210 lymphocytic leukemia cells into BDF1 recipients. At 12-h intervals for the first 2 days after tumor-cell injection and at 6-h intervals thereafter, animals were observed and scored for each parameter. When death was imminent, animals were euthanized by inhalation of methoxyflurane followed by decapitation. Changes in physical and behavioral characteristics then were correlated with the end-point of death. Changes occurred in the mice approximately 7 days after tumor cell inoculation and 24 h before death. The earliest of these signs was hunched posture, followed by one or more other characteristics including decreased activity, increased facial swelling, ears in backward position, abdominal swelling, squinting eyes, and labored breathing. From these data, we were able to develop criteria for early euthanasia. Use of these intermediate end-points likely will substantially reduce the stress on the animals without compromising scientific outcomes in experiments using this or related preclinical models of cancer.

Temporal effects of gamma interferon deficiency on the course of Friend retrovirus infection in mice.

The current studies demonstrate complex and seemingly contradictory effects by gamma interferon (IFN-gamma) on Friend virus (FV) infection. Both temporal and tissue-specific effects were observed. During the first week of infection, IFN-gamma-deficiency caused increased levels of FV infection in multiple tissues. Surprisingly, however, by 2 weeks postinfection, IFN-gamma-deficient mice had significantly lower levels of infection in both the spleen and bone marrow compared to wild-type mice. The rapid reduction of virus in the IFN-gamma-deficient mice correlated with a more rapid virus-neutralizing antibody response than was observed in the wild-type mice. Furthermore, the virus-neutralizing antibody response in wild-type mice could be accelerated by ablation of their IFN-gamma response. Although the IFN-gamma-deficient mice developed an accelerated virus-neutralizing antibody response, they did not class-switch to immunoglobulin G class immunoglobulins nor could they maintain long-term virus-neutralizing antibody titers. Eventually, all of the IFN-gamma-deficient mice failed to keep persistent virus in check and developed fatal FV-induced erythroleukemia.

Disruption of hematopoiesis and thymopoiesis in the early premalignant stages of infection with SL3-3 murine leukemia virus.

A time course analysis of SL3-3 murine leukemia virus (SL3) infection in thymus and bone marrow of NIH/Swiss mice was performed to assess changes that occur during the early stages of progression to lymphoma. Virus was detectable in thymocytes, bone marrow, and spleen as early as 1 to 2 weeks postinoculation (p.i.). In bone marrow, virus infection was detected predominantly in immature myeloid or granulocytic cells. Flow cytometry revealed significant reductions of the Ter-119(+) and Mac-1(+) populations, and significant expansions of the Gr-1(+) and CD34(+) populations, between 2 and 4 weeks p.i. Analysis of colony-forming potential confirmed these findings. In the thymus, SL3 replication was associated with significant disruption in thymocyte subpopulation distribution between 4 and 7 weeks p.i. A significant thymic regression was observed just prior to the clonal outgrowth of tumor cells. Proviral long terminal repeats (LTRs) with increasing numbers of enhancer repeats were observed to accumulate exclusively in the thymus during the first 8 weeks p.i. Observations were compared to the early stages of infection with a virtually nonpathogenic SL3 mutant, termed SL3DeltaMyb5, which was shown by real-time PCR to be replication competent. Comparison of SL3 with SL3DeltaMyb5 implicated certain premalignant changes in tumorigenesis, including (i) increased proportions of Gr-1(+) and CD34(+) bone marrow progenitors, (ii) a significant increase in the proportion of CD4(-) CD8(-) thymocytes, (iii) thymic regression prior to tumor outgrowth, and (iv) accumulation of LTR enhancer variants. A model in which disrupted bone marrow hematopoiesis and thymopoiesis contribute to the development of lymphoma in the SL3-infected animal is discussed.