HiHiMap: single-cell quantitation of histones and histone posttranslational modifications across the cell cycle by high-throughput imaging.

We describe High-throughput Histone Mapping (HiHiMap), a high-throughput imaging method to measure histones and histone posttranslational modifications (PTMs) in single cells. HiHiMap uses imaging-based quantification of DNA and cyclin A to stage individual cells in the cell cycle to determine the levels of histones or histone PTMs in each stage of the cell cycle. As proof of principle, we apply HiHiMap to measure the level of 21 core histones, histone variants, and PTMs in primary, immortalized, and transformed cells. We identify several histone modifications associated with oncogenic transformation. HiHiMap allows the rapid, high-throughput study of histones and histone PTMs across the cell cycle and the study of subpopulations of cells.

Common features of chromatin in aging and cancer: cause or coincidence?

Age is a major risk factor for cancer. Alterations in DNA methylation, histone modifications, chromatin structure, and epigenetic regulatory mechanisms are prominent hallmarks of both the aging process and cancer. Intriguingly--or possibly coincidentally--several chromatin features are common between aging and cancer. Here we ask whether, and if so how, aging-associated chromatin modifications contribute to tumor susceptibility and tumorigenesis.

HTLV-1 and leukemogenesis: virus-cell interactions in the development of adult T-cell leukemia.

Human T-cell lymphotropic virus type 1 (HTLV-1) was originally discovered in the early 1980s. It is the first retrovirus to be unambiguously linked causally to a human cancer. HTLV-1 currently infects approximately 20 million people worldwide. In this chapter, we review progress made over the last 30 years in our understanding of HTLV-1 infection, replication, gene expression, and cellular transformation.

Mosaicism of HTLV-1 5' LTR CpG methylation in the absence of malignancy.

Viral expression varies widely between untransformed HTLV-1 positive clones derived from infected individuals without malignancy. Here we show that, in the absence of malignancy, 68% of HTLV-1 positive clones carry deleted (10%) or methylated (58%) 5' LTR. These changes were found to contribute to the fluctuation of viral expression between clones. 5' LTR deletions strongly impaired tax expression and thereby clonal expansion, telomere homeostasis, and genetic instability. The effects of CpG methylation on viral transcription were qualitatively and quantitatively different from those of LTR deletions and preserved the preleukemic features of HTLV-1(+)CD4(+) clones. 5' LTR methylation varied not only between clones but also between cells belonging to the same clones, between distinct integrated sequences within the same cells, and between CpG dyads along the 5' LTR. Such distributions suggest that a dynamic methylation spectrum might help sustain persistent infection.

The cellular autophagy pathway modulates human T-cell leukemia virus type 1 replication.

Autophagy, a general homeostatic process for degradation of cytosolic proteins or organelles, has been reported to modulate the replication of many viruses. The role of autophagy in human T-cell leukemia virus type 1 (HTLV-1) replication has, however, been uncharacterized. Here, we report that HTLV-1 infection increases the accumulation of autophagosomes and that this accumulation increases HTLV-1 production. We found that the HTLV-1 Tax protein increases cellular autophagosome accumulation by acting to block the fusion of autophagosomes to lysosomes, preventing the degradation of the former by the latter. Interestingly, the inhibition of cellular autophagosome-lysosome fusion using bafilomycin A increased the stability of the Tax protein, suggesting that cellular degradation of Tax occurs in part through autophagy. Our current findings indicate that by interrupting the cell's autophagic process, Tax exerts a positive feedback on its own stability.

Wip1 and p53 contribute to HTLV-1 Tax-induced tumorigenesis.

BACKGROUND: Human T-cell Leukemia Virus type 1 (HTLV-1) infects 20 million individuals world-wide and causes Adult T-cell Leukemia/Lymphoma (ATLL), a highly aggressive T-cell cancer. ATLL is refractory to treatment with conventional chemotherapy and fewer than 10% of afflicted individuals survive more than 5 years after diagnosis. HTLV-1 encodes a viral oncoprotein, Tax, that functions in transforming virus-infected T-cells into leukemic cells. All ATLL cases are believed to have reduced p53 activity although only a minority of ATLLs have genetic mutations in their p53 gene. It has been suggested that p53 function is inactivated by the Tax protein. RESULTS: Using genetically altered mice, we report here that Tax expression does not achieve a functional equivalence of p53 inactivation as that seen with genetic mutation of p53 (i.e. a p53 -/- genotype). Thus, we find statistically significant differences in tumorigenesis between Tax+p53 +/+ versus Tax+p53 -/- mice. We also find a role contributed by the cellular Wip1 phosphatase protein in tumor formation in Tax transgenic mice. Notably, Tax+Wip1 -/- mice show statistically significant reduced prevalence of tumorigenesis compared to Tax+Wip1 +/+ counterparts. CONCLUSIONS: Our findings provide new insights into contributions by p53 and Wip1 in the in vivo oncogenesis of Tax-induced tumors in mice.

The importance of ubiquitination and sumoylation on the transforming activity of HTLV Tax-1 and Tax-2.

Human T-cell Leukemia Virus type 1 (HTLV-1) and 2 (HTLV-2) are two closely related human retroviruses. HTLV-1 is associated with an aggressive Adult T-cell Leukemia (ATL) while there is no evidence for an association of HTLV-2 with any human malignancies. The two viruses encode transactivator proteins, Tax-1 and Tax-2 respectively. In ATL, Tax-1 is thought to play a central role in the transformation of a normal T-cell into a leukemic cell; however, it has not been entirely clear how post-translational modifications of Tax-1 influence its transforming activity. Here, we discuss three recent papers that report on the ubiquitination and sumoylation of Tax-1 and Tax-2. We comment on their divergent findings implicating the importance (or lack of importance) of these modifications and other events on Tax activation of NF-κB as related to cellular transformation.

HTLV-1 positive and negative T cells cloned from infected individuals display telomerase and telomere genes deregulation that predominate in activated but untransformed CD4+ T cells.

Untransformed HTLV-1 positive CD4(+) cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV-1 positive CD4(+) cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression, whereas their increase in telomerase activity is about 20 times lower than that of their uninfected counterpart. Activated HTLV-1 positive CD4(+) but not uninfected CD4(+) or CD8(+) clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA-PKc and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4(+) cells. In conclusion HTLV-1-dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression.

Telomere deregulations possess cytogenetic, phenotype, and prognostic specificities in acute leukemias.

OBJECTIVE: Telomeres are protected by tightly regulated factors and elongated by telomerase. Short and/or deprotected chromosomes are recombinogenic and thereby cancer prone. MATERIALS AND METHODS: Together with the quantification of telomerase activity (TA), measuring telomere length (TL) and expression of the genes that govern telomere protection and elongation are useful for assessing telomere homeostasis. RESULTS: By these means we demonstrate that TL, hTERT, and TA are in the order acute myelogenous leukemia (AML) > T-cell acute lymphoblastic leukemia (T-ALL) > B-cell acute lymphoblastic leukemia (B-ALL) > T-ALL > AML, and B-ALL > AML > T-ALL. AML0 and AML3 display the lowest amounts of hTERT transcripts, and ALL and AML cells with cytogenetic abnormalities possess the shortest telomeres. hTERT expression includes phenotype-specific RNA maturation and correlates with TA but not with TL. A wide ratio of TA to hTERT expression between leukemia subtypes suggests phenotype-specific hTERT post-transcriptional deregulations. B- and T-ALL overexpress Ku70 and Pinx1, T-ALL PTOP and RAP1, and B-ALL TRF2, the expression of which is significantly higher in cases with abnormal karyotype. hTERT transcription and TL correlate with response to intensive chemotherapy, and hTERT and RAD50 are independent prognostic factors for survival. CONCLUSIONS: Each leukemia subtype possesses specific telomere dysregulations that rely on phenotype, karyotype, response to treatment, and survival.

Clonal expansion of HTLV-1 positive CD8+ cells relies on cIAP-2 but not on c-FLIP expression.

Here we investigate the mechanisms by which HTLV-1 infection prevents the cell death of CD8(+) T cells in vivo. We show that upon natural infection, cloned CD8(+) but not CD4(+) cells from patients without malignancy become resistant to Fas-mediated cell death and acquire an antiapoptotic transcriptome that includes the overexpression of cIAP-2 and c-FLIP(L). CD8(+) lymphocyte-restricted cIAP-2 overexpression correlates with resistance to Fas-mediated apoptosis and depends on tax expression via NF-KappaB. In contrast, in the same CD8(+) cells, the HTLV-1-dependent overexpression of c-FLIP(L) does not correlate with resistance to Fas-mediated cell death nor with tax expression. In the present model, infected CD8(+) clones are the only cell subtype in which cIAP-2 expression correlates with resistance to cell death. These results support a role for Tax-dependent cIAP-2 expression in preventing the death of naturally infected CD8(+) cells and thereby in their clonal expansion in vivo.

Tax gene expression and cell cycling but not cell death are selected during HTLV-1 infection in vivo.

BACKGROUND: Adult T cell leukemia results from the malignant transformation of a CD4+ lymphoid clone carrying an integrated HTLV-1 provirus that has undergone several oncogenic events over a 30-60 year period of persistent clonal expansion. Both CD4+ and CD8+ lymphocytes are infected in vivo; their expansion relies on CD4+ cell cycling and on the prevention of CD8+ cell death. Cloned infected CD4+ but not CD8+ T cells from patients without malignancy also add up nuclear and mitotic defects typical of genetic instability related to the expression of the virus-encoded oncogene tax. HTLV-1 expression is cancer-prone in vitro, but in vivo numerous selection forces act to maintain T cell homeostasis and are possibly involved in clonal selection. RESULTS: Here we demonstrate that the HTLV-1 associated CD4+ preleukemic phenotype and the specific patterns of CD4+ and CD8+ clonal expansion are in vivo selected processes. By comparing the effects of recent (1 month) experimental infections performed in vitro and those observed in cloned T cells from patients infected for >6-26 years, we found that in chronically HTLV-1 infected individuals, HTLV-1 positive clones are selected for tax expression. In vivo, infected CD4+ cells are positively selected for cell cycling whereas infected CD8+ cells and uninfected CD4+ cells are negatively selected for the same processes. In contrast, the known HTLV-1-dependent prevention of CD8+ T cell death pertains to both in vivo and in vitro infected cells. CONCLUSIONS: Therefore, virus-cell interactions alone are not sufficient to initiate early leukemogenesis in vivo.

Clonal expansion of HTLV-1 infected cells depends on the CD4 versus CD8 phenotype.

As other deltaretroviruses HTLV-1 replication in vivo includes a first short step of reverse transcription that is followed by the persistent clonal expansion of infected cells. In vivo these cells include the CD4+ and CD8+ lymphocytes yet the virus induces adult T cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Cloned infected cells from individuals without malignancy possess a dramatic increase in spontaneous proliferation, which predominated with CD8+ lymphocytes and depends on the amount of tax mRNA. In fact, the clonal expansion of HTLV-1 positive CD8+ and CD4+ lymphocytes relies on two distinct mechanisms: infection prevented cell death in the former whereas recruiting the latter into the cell cycle. Furthermore infected tax-expressing CD4+ lymphocytes cumulate cellular defects characteristic of genetic instability. Therefore, HTLV-1 infection establishes a preleukemic phenotype that is restricted to CD4+ infected clones. Investigating the mechanisms underlying apoptosis, cell cycling and DNA repair in cloned cells from naturally infected individuals will permit to deciphering the molecular pathogenesis of HTLV-1 infection.