AKR leukemogenesis: identification and biological significance of thymic lymphoma receptors for AKR retroviruses.

We have previously demonstrated that in vitro cell lines of mouse thymic lymphomas express surface receptors specific for the retrovirus that induced them. This study extends these observations to an analysis of receptor-bearing cells in the preleukemic and leukemic phases of spontaneous AKR thymic lymphomagenesis. AKR mice regularly begin expressing N-tropic retroviruses (as assayed on NIH fibroblasts by the XC plaque assay) in several tissues early in life; thymic lymphocytes also express these viruses, but are not autonomously transformed. Later thymic lymphomas emerge which are capable of metastasizing in the host of origin or transplanting leukemias into syngeneic hosts. Just prior to the appearance of thymic lymphomas, these mice also begin producing xenotropic retroviruses [as assayed in xenogeneic (For example, mink) fibroblasts], and concomitant with the appearance of the leukemias is the appearance of "recombinant" retroviruses which cause mink fibroblast foci (MCF); these viruses express elements of both N- and X-tropic virus envelopes and N-tropic viral gene products in their cores. Spontaneous AKR leukemias also produce other retroviruses which do not cause XC plaques or mink fibroblast foci; these are called SL viruses. The subject of this study was to test whether in vivo thymocytes in the preleukemic and leukemic periods also bear receptors specific for N-tropic, recombinant MCF and SL AKR retroviruses. We demonstrated that each spontaneous thymic lymphoma does bear receptors that bind viruses produced by the lymphomas and MCF-247 to a high degree and that bind N-ecotropic AKR retroviruses less well. Thymic lymphocytes predominating in the preleukemic period do not express detectable levels of receptors for either of the viruses. In some mice, receptor-positive cells co-exist with receptor-negative cells; only the receptor-positive cells are capable of transplanting leukemia to syngeneic hosts. We conclude that the presence of specific cell surface receptors for lymphoma cell-produced and recombinant AKR retroviruses is a marker for leukemia in these hosts.     

The nucleolar apparatus of the lymphoid cells in AKR mice at different stages of leukemogenesis

Morphological features of nuclear apparatus in mice leukemic cells of the peripheral blood, lymph nodes, thymus, bone marrow and spleen were studied. These cells were taken from 12-months-old AKR mice. Enlarged percentage of cells with nucleolar lipid component in thymus characterizes preleukemic state for 6-months-old AKR line mice.     

Changes in thymocyte proliferation at different stages of viral leukemogenesis in AKR mice

Proliferation in total populations of thymocytes from control AKR mice or AKR mice injected intrathymically with MCF 69L1 virus was measured by flow cytometry of acridine orange-stained cells. Cell sorting experiments showed that the majority subpopulations of small cortical and medullary thymocytes in control mice were noncycling and were predominantly in the Go phase of the cell cycle. Of the 15 to 20% cycling thymic lymphoblasts, approximately 50% were in the G1 phase, 35% were in the S phase, and 15% were in the G2 + M phases of the cell cycle. Cycling cells appeared to consist of a major subpopulation with low RNA content and a minority subpopulation with high RNA content. In virus-injected mice, no changes in cell cycling were observed at stage I of leukemogenesis (30 to 40 days postinjection), at which time infection of thymocytes by MCF virus is maximum and constant but no clonality is evident. Thus, MCF virus infection of thymocytes per se does not appear to alter cell proliferation. Increased cell cycling and a shift in cell cycle distribution to more cells in G1 was observed at stage II of leukemogenesis (50 to 60 days postinjection), at which time a clonally expanded cell population is known to emerge in thymuses of injected mice. Acridine orange staining resolved these novel cycling cells from subpopulations of normal thymic lymphoblasts on the basis of intermediate RNA content. The transition from stage II to stage III (50 to 60 days postinjection) was accompanied by the outgrowth of a major cycling population with a distinct, often increased, RNA content. As a result, the residual "normal" background of cycling cells often observed in stage II was markedly reduced or completely absent by stage III. Populations of cycling blasts from mice with frank leukemia differed from those at stage III by a variability in mean RNA content and in cell cycle distribution indicative of individual tumor heterogeneity. In addition, thymomas often contained multiple populations of cycling blasts that could be resolved by their discrete RNA distributions. Simultaneous staining of DNA and RNA by acridine orange appears particularly well-suited for studying a heterogeneous population of cycling and noncycling cells represented by mouse thymus. This method has permitted a rapid and quantitative analysis of cell cycle parameters at progressive stages of viral leukemogenesis in AKR mice.     

Macrophages are the first thymic cells to express polytropic retrovirus in AKR mouse leukemogenesis.

AKR mice spontaneously develop T-cell leukemias in the thymus late in the first year of life. These neoplasms arise following the appearance in the thymus of a recombinant retrovirus but can be prevented by thymectomy, indicating a role for both virus and elements of the thymic microenvironment in leukemogenesis. The intrathymic appearance of recombinant retrovirus was examined at ages leading up to leukemogenesis in order to identify and characterize the microenvironments in which the virus is first expressed. A stromal cell, the macrophage, was found to be the first thymic element to produce detectable levels of recombinant retrovirus, approximately 12 weeks before thymocytes. This observation provides a mechanism to reconcile viral leukemogenesis with the requirement for an intact thymus. Thus, a nonlymphoid cell, the macrophage, may play a critical role in the development of lymphoid neoplasia.     

Modulation of fibronectin synthesis and fibronectin binding during transformation and differentiation of mouse AKR fibroblasts

In previous studies it was shown that transformation of AKR fibroblasts with 3-methylcholanthrene was associated with a loss of surface fibronectin and that induction of differentiation of the transformed cells with N,N-dimethylformamide (DMF) was associated with reacquisition of surface fibronectin (Chakrabarty et al., J. Cell. Physiol. 133:415, 1987). It is shown in the present study that changes in surface fibronectin reflect altered fibronectin synthesis and altered fibronectin binding. Both the nontransformed cells (AKR-2B) and their transformed counterparts (AKR-MCA) bound 125I-fibronectin in a receptor-like fashion, but the AKR-MCA cells had only 20% of the receptors found on the AKR-2B cells. Whole cell extracts prepared from the AKR-2B cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions were examined for 125I-fibronectin binding. Under these conditions, the majority of binding occurred to moieties with molecular weights of 180 kD, 150 kD, and 97 kD. Binding to similar moieties on the AKR-MCA cells was virtually absent but occurred rapidly after treatment with DMF. The appearance of these moieties paralleled the acquisition of 125I-fibronectin binding activity by whole cells. Antibodies to the fibronectin receptor isolated from human placenta reacted with the DMF-sensitive moieties in immunoblot assays. Both the appearance of the fibronectin binding moieties and the acquisition of 125I-fibronectin binding activity by whole cells occurred within 6 hr of DMF treatment and increased over the subsequent 4 day period. The time course of these events paralleled closely the time course for induction of fibronectin biosynthesis by DMF. These changes in fibronectin binding and fibronectin production were associated with alterations in cell-substrate adhesion. The AKR-2B cells rapidly attached and spread on bovine serum albumin-coated dishes and on fibronectin-coated dishes, whereas the AKR-MCA cells were less adhesive on both substrates. Capacity to attach and spread was regained concomitantly with the induction of fibronectin binding and fibronectin production. Adhesion on both substrates was partially inhibited by antibodies to the fibronectin receptor and by RGDS. These studies suggest that fibronectin production and fibronectin binding are coregulated in AKR fibroblasts and that they function together to bring about changes in cell-substrate adhesion.     

Protein patterns during final differentiation of some rat organs

Development Genes and Evolution -     

Changing patterns of placental hexokinase isozymes during the course of gestation.

Hexokinase, an enzyme that is capable of regulating the entry of glucose into metabolic sequences, is known to exist in four isozyme forms in a variety of mammalian tissues. Each of these isozymes possesses different kinetic properties, suggesting that they may serve in different regulatory capacities. In addition, the proportions of the four isozymes are characteristically different for various differentiated tissues, presumably reflecting different metabolic capabilities of the tissues.Extracts of rabbit and human placentas from early gestational age and term pregnancies were chromatographed and assayed for hexokinase activity. Four peaks of activity were observed. Elution positions of the four placental hexokinase isozymes were comparable to those from liver; however, the relative proportions were considerably different. In both rabbit and human placentas, the proportion of Type I hexokinase increased during gestation and Type III decreased, while Types II and IV remained essentially unchanged. Implications of the gestational changes for placental carbohydrate metabolism are discussed.     

Analysis of tumor-associated alkyldiacylglycerols and other lipids during radiation-induced thymic leukemogenesis.

Lipid composition of thymuses investigated during the development of thymic leukemogenesis induced by exposing C57BL/6J mice to gamma radiation led to the following conclusions. 1. Alkyldiacylglycerols, a class of lipids that are generally elevated in most neoplastic tissues, occurred only in small quantities (less than 1% of the total lipids) in the thymuses of both control and irradiated mice. However, we found a 3- to 8-fold increase in this fraction in thymic tumors of mice at 5 mo after irradiation when compared to controls of similar age. However, the small quantity of this lipid class in thymic leukemia and the fact that similar levels were found in some samples of involuted thymuses of mice 3 days after irradiation, suggests to us that the level of alkyldiacylglycerols is not sufficiently specific or sensitive for detecting early stages of thymic leukemogenesis. 2. Thymuses 3 days after irradiation and leukemic thymuses contain 2- to 3-fold greater quantities of cholesterol esters than control thymuses. No major differences were found in the distribution of acyl moieties in the cholesterol esters of the various thymus samples from the same aged mice except that in thymic tumors the quantity of 18:1 esters was increased by about 25% over that of the controls. The apparent lack of specificity of increased cholesterol esters for neoplasia indicates that its measurement would not provide a suitable indicator of early neoplastic transformation. 3. Acyl composition of the triacylglycerols of thymuses revealed an increase in the 18:1 and a decrease in the 18:2 acids at 3 days after irradiation when compared to the same aged controls. However, thymic tumors occurring at 5 mo after irradiation contained less 14:0, 16:0, and 16:1 acids and increased amounts of 18:1 and 18:2 acids esterified as triacylglycerols compared to controls. The fatty acid distribution in the phospholipid fraction of thymuses was not altered by the appearance of leukemia.     

Glucocorticoid receptors and corticosensitivity of human thymocytes at discrete stages of intrathymic differentiation

Human thymus is composed of several discrete compartments. Stage III thymocytes, located mainly in the medulla, stain brightly with anti-T3 monoclonal antibody; stage II thymocytes, located in the cortex, are T3- but react with T6 antibodies. The earliest identifiable intrathymic cell (stage I) expresses the sheep erythrocyte glycoprotein T11 but not T6 or T3 antigens. Within the thymus a phenotypically heterogeneous pool of proliferating lymphoblasts is present. This capacity to proliferate without in vitro activation is mainly attributable to thymocytes unable to respond to mitogens and expressing the cortical T6 marker. Both T3+ and T3-T6- cells respond to mitogen. However, in order to exhibit maximal proliferative responses, T3+ but not T3-T6- thymocytes require the addition of exogenous IL 2. Thymocyte subsets at distinct stages of intrathymic differentiation were then analyzed for glucocorticoid (GC) receptor content by using a whole cell assay with 3H-triamcinolone acetonide as tracer. The least mature T3-T6- thymocyte subset contained the highest levels of GC receptors . T3+ thymocytes exhibited a receptor content higher than that found in T6+ cells and similar to that reported for peripheral blood lymphocytes. Apart from the number, the GC receptor sites in all thymocyte subsets were similar in their affinities, kinetic characteristics, specificity for steroids, and ability to undergo translocation from cytoplasm to nucleus, and they behave in all these respects like binding sites of GC receptors in lymphoid and other cells. Independently of both phenotype and GC receptor content, all in vivo activated thymocytes (i.e., spontaneously proliferating cells) were similarly sensitive to the steroid inhibitory action in vitro. Both in the presence and in the absence of exogenous IL 1 or IL 2, the PHA-induced mitogenesis of T3-T6- cells was less inhibited by GC than that of T3+ thymocytes. Exogenous IL 1 and IL 2 were equally effective in removing, although not completely, the GC inhibition on T3-T6- proliferative responses to PHA. Relative to T3+ cell mitogenesis, only exogenous IL 2 was able to antagonize the steroid inhibitory action. The capacity observed in vitro of GC to differentially affect the proliferative potential or the cell viability of thymocytes belonging to functionally distinct subsets suggests that these hormones could regulate the intrathymic maturative pathways. Finally, although at present the physiologic relevance of the highest expression of GC receptors in intrathymic precursor cells remains unclear, the receptor density may be considered a marker of differentiation for the T lymphoid lineage.     

Peroxidases during the course of callusing and organ differentiation from root explants ofCichorium intybus

Growth ofCichorium intybus root explants was accompanied by an important increase of fresh mass during the course of callusing and rooting. The absence of glucose in callus forming medium was compensated for by hydrolysis of storage carbohydrates of the tissues, inducing a decrease in dry mass. Protein content showed similar slight variations in explants during the course of budding and callusing, whereas an important increase of protein content was found during the first 48 h in explants cultured on root forming medium. Specific increase of soluble peroxidase activity during bud and root neoformation was found. A peak of peroxidase activity, proceeding the organ emergence, was always observed. This peak occurred earlier in bud forming than in root forming explants. Conversely, during callusing, the bulk peroxidase activity showed only weak variations, and no peak was detectable. Moreover one basic isoperoxidase was missing in these explants after a 6-d culture, whereas in both root and bud forming explants the isoperoxidase patterns were very similar. When explants growing on differentiation media were in darkness a supplementary basic isoperoxidase with the highest electrophoretic mobility was revealed.     

Protein synthetic patterns during differentiation of imaginal discs in vitro.

Leg, wing, and eye-antennal imaginal discs of Drosophila melanogaster were obtained by dissection from late-third instar larvae and cultured in vitro in Schneider's medium containing β-ecdysone. Differentiation of adult structures was obtained in more than 90% of all cultures. Differentiation was somewhat slower than normal in vivo development, but synchronous, repeatable, and reasonably completed structures were obtained. Our initial efforts at analyzing the molecular events of imaginal disc differentiation in culture have been to study the protein synthetic pattern which occurs throughout the culture period. Discs were pulse labeled with [³⁵S]methionine, and the proteins were separated by SDS-gel electrophoresis. Analysis of the synthetic pattern was done by autoradiography of these gels using X-ray film. In all three disc types, pronounced changes in protein synthetic patterns occurred throughout the culture period. These changes appeared to be under strict temporal control. Although disc-specific differences could be seen, a comparison of the three discs types revealed a striking similarity in the changes which occurred in the patterns of protein synthesis during the 5-day culture period. In general, the protein synthetic patterns of different imaginal discs at the same period during differentiation showed greater similarities than the patterns of a single disc type at different periods. These results are consistent with a view of differentiation as a tightly controlled program of gene activation and deactivation operating throughout the differentiation process.     

T-cell subsets in the murine thymus during chemically induced leukemogenesis

T cell leukemias were induced in BDF 1 mice by methylnitrosourea (MNU). The phenotype of the leukemic cells is Thy1.2 +, PNA-, TdT+, TL+ and heterogeneous with respect to Lyt-1 and Lyt-2. About 70% of the leukemias have elevated amounts of gp70. During latency period of at least 9 + 12 weeks an early reduction in the various thymic cells and the CFU-S is observed, with almost complete recovery. Later PNA+ cells are reduced. Hydrocortisone treatment delays or enhances leukemogenesis, dependent on the time interval between hydrocortisone and MNU. Some mice show elevated amounts of gp70 in their bone marrow 2--3 weeks after MNU. The problem of target cells in the bone marrow and the thymus is discussed.     

Deciphering the heterogeneity in DNA methylation patterns during stem cell differentiation and reprogramming

Background

Human induced pluripotent stem cells (iPSCs) have a wide range of applications throughout the fields of basic research, disease modeling and drug screening. Epigenetic instable iPSCs with aberrant DNA methylation may divide and differentiate into cancer cells. Unfortunately, little effort has been taken to compare the epigenetic variation in iPSCs with that in differentiated cells. Here, we developed an analytical procedure to decipher the DNA methylation heterogeneity of mixed cells and further exploited it to quantitatively assess the DNA methylation variation in the methylomes of adipose-derived stem cells (ADS), mature adipocytes differentiated from ADS cells (ADS-adipose) and iPSCs reprogrammed from ADS cells (ADS-iPSCs).

Results

We observed that the degree of DNA methylation variation varies across distinct genomic regions with promoter and 5’UTR regions exhibiting low methylation variation and Satellite showing high methylation variation. Compared with differentiated cells, ADS-iPSCs possess globally decreased methylation variation, in particular in repetitive elements. Interestingly, DNA methylation variation decreases in promoter regions during differentiation but increases during reprogramming. Methylation variation in promoter regions is negatively correlated with gene expression. In addition, genes showing a bipolar methylation pattern, with both completely methylated and completely unmethylated reads, are related to the carbohydrate metabolic process, cellular development, cellular growth, proliferation, etc.

Conclusions

This study delivers a way to detect cell-subset specific methylation genes in a mixed cell population and provides a better understanding of methylation dynamics during stem cell differentiation and reprogramming.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-978) contains supplementary material, which is available to authorized users.     

Correlation of cell division and specific protein production during the course of lymphoid cell differentiation

Mice were immunized with horseradish peroxidase and injected with [³H] thymidine. Popliteal lymph node cells were submitted to electron microscopic immunocytochemistry and high resolution autoradiography in order to correlate antibody production and ability to undergo cell division at various stages of lymphoid cell differentiation. Antibody synthesis started in blast cells and increased steadily until the mature plasma cell stage was reached. Thymidine incorporation was highest in blastoid cells and decreased continuously afterwards. Chromatin dispersion was found to be paralleled by thymidine incorporation. This observation and data of other authors seem to indicate that chromatin dispersion is a prerequisite for replication. Almost all mature plasma cells were devoid of thymidine incorporation. This confirms that they are end cells apparently unable to divide.