Identification of inflections in T-cell counts among HIV-1-infected individuals and relationship with progression to clinical AIDS

Studies of circulating T (CD3+) lymphocytes have shown that on a population basis T-cell numbers remain stable for many years after HIV-1 infection (blind T-cell homeostasis), but decline rapidly beginning approximately 1.5–2.5 years before the onset of clinical AIDS. We derived a general method for defining the loss of homeostasis on the individual level and for determining the prevalence of homeostasis loss according to HIV status and the occurrence of AIDS in more than 5,000 men enrolled in the Multicenter AIDS Cohort Study. We used a segmented regression model for log10 CD3+ cell counts that included separate T-cell trajectories before and after a time (the T-cell inflection point) where the loss of T-cell homeostasis was most likely to have occurred. The average slope of CD3+ lymphocyte counts before the inflection point was close to zero for HIV− and HIV+ men, consistent with blind T-cell homeostasis. After the inflection point, the HIV+ individuals who developed AIDS generally showed a dramatic decline in CD3+ cell counts relative to HIV− men and HIV+ men not developing AIDS. A CD3+ cell decline of greater than 10 percent per year was present in 77% of HIV+ men developing AIDS but in only 23% of HIV+ men with no onset of AIDS. Our findings at the individual level support the blind T-cell homeostasis hypothesis and provide strong evidence that the loss of homeostasis is an important mechanism in the pathogenesis of the severe immunodeficiency that characterizes the late stages of HIV infection.

Interferon-γ impacts at multiple points during the progression of autoimmune diabetes

The role of interferon-γ in autoimmune diabetes was assessed by breeding a null mutation of the interferon-γ receptor α chain into the nonobese diabetic mouse strain, as well as into a simplified T cell receptor transgenic model of diabetes. In contrast to a previous report on abrogation of the interferon-γ gene, mutation of the gene encoding its receptor led to drastic effects on disease in both mouse lines. Nonobese diabetic mice showed a marked inhibition of insulitis—both the kinetics and penetrance—and no signs of diabetes; the transgenic model exhibited near-normal insulitis, but this never evolved into diabetes, either spontaneously or after experimental provocation. This failure could not be explained by perturbations in the ratio of T helper cell phenotypes; rather, it reflected a defect in antigen-presenting cells or in the islet β cell targets.

Regulation of Cell Cycle Progression by Swe1p and Hog1p Following Hypertonic Stress

Exposure of yeast cells to an increase in external osmolarity induces a temporary growth arrest. Recovery from this stress is mediated by the accumulation of intracellular glycerol and the transcription of several stress response genes. Increased external osmolarity causes a transient accumulation of 1N and 2N cells and a concomitant depletion of S phase cells. Hypertonic stress triggers a cell cycle delay in G2 phase cells that appears distinct from the morphogenesis checkpoint, which operates in early S phase cells. Hypertonic stress causes a decrease in CLB2 mRNA, phosphorylation of Cdc28p, and inhibition of Clb2p-Cdc28p kinase activity, whereas Clb2 protein levels are unaffected. Like the morphogenesis checkpoint, the osmotic stress-induced G2 delay is dependent upon the kinase Swe1p, but is not tightly correlated with inhibition of Clb2p-Cdc28p kinase activity. Thus, deletion of SWE1 does not prevent the hypertonic stress-induced inhibition of Clb2p-Cdc28p kinase activity. Mutation of the Swe1p phosphorylation site on Cdc28p (Y19) does not fully eliminate the Swe1p-dependent cell cycle delay, suggesting that Swe1p may have functions independent of Cdc28p phosphorylation. Conversely, deletion of the mitogen-activated protein kinase HOG1 does prevent Clb2p-Cdc28p inhibition by hypertonic stress, but does not block Cdc28p phosphorylation or alleviate the cell cycle delay. However, Hog1p does contribute to proper nuclear segregation after hypertonic stress in cells that lack Swe1p. These results suggest a hypertonic stress-induced cell cycle delay in G2 phase that is mediated in a novel way by Swe1p in cooperation with Hog1p.

Allelic loss on chromosome band 18p11.3 occurs early and reveals heterogeneity in breast cancer progression

We examined the stage specificity and heterogeneity of 18p11 alterations in a series of tumors representing 96 microdissected samples. Significant loss of heterozygosity (LOH) (63%) was found, with 56% occurring early in ductal carcinoma in situ. Although most cases indicated LOH was clonally inherited, heterogeneity for 18p LOH occurred in 27% of tumors. When compared with other LOH data, 18p LOH was found in conjunction with allelic deletion on 3p, 9p, 17p and 17q, while 13q, 16q, and 11p were less frequently associated. These analyses suggest chromosome 18p11 alteration is a common and early event in breast disease.

Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK+ yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8–9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 ‘early’ origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.

PEA3 sites within the progression elevated gene-3 (PEG-3) promoter and mitogen-activated protein kinase contribute to differential PEG-3 expression in Ha-ras and v-raf oncogene transformed rat embryo cells

Transformation of normal cloned rat embryo fibroblast (CREF) cells with cellular oncogenes results in acquisition of anchorage-independent growth and oncogenic potential in nude mice. These cellular changes correlate with an induction in the expression of a cancer progression-promoting gene, progression elevated gene-3 (PEG-3). To define the mechanism of activation of PEG-3 as a function of transformation by the Ha-ras and v-raf oncogenes, evaluations of the signaling and transcriptional regulation of the ~2.0 kb promoter region of the PEG-3 gene, PEG-Prom, was undertaken. The full-length and various mutated regions of the PEG-Prom were linked to a luciferase reporter construct and tested for promoter activity in CREF and oncogene-transformed CREF cells. An analysis was also performed using CREF cells doubly transformed with Ha-ras and the Ha-ras specific suppressor gene Krev-1, which inhibits the transformed phenotype in vitro. These assays document an association between expression of the transcription regulator PEA3 and PEG-3. The levels of PEA3 and PEG-3 RNA and proteins are elevated in the oncogenically transformed CREF cells, and reduced in transformation and tumorigenic suppressed Ha-ras/Krev-1 doubly transformed CREF cells. Enhanced tumorigenic behavior, PEG-3 promoter function and PEG-3 expression in Ha-ras transformed cells were all dependent upon increased activity within the mitogen-activated protein kinase (MAPK) pathway. Electrophoretic mobility shift assays and DNase I footprinting experiments indicate that PEA3 binds to sites within the PEG-Prom in transformed rodent cells in an area adjacent to the TATA box in a MAPK-dependent fashion. These findings demonstrate an association between Ha-ras and v-raf transformation of CREF cells with elevated PEA3 and PEG-3 expression, and they implicate MAPK signaling via PEA3 as a signaling cascade involved in activation of the PEG-Prom.

BimD/SPO76 is at the interface of cell cycle progression, chromosome morphogenesis, and recombination

BIMD of Aspergillus nidulans belongs to a highly conserved protein family implicated, in filamentous fungi, in sister-chromatid cohesion and DNA repair. We show here that BIMD is chromosome associated at all stages, except from late prophase through anaphase, during mitosis and meiosis, and is involved in several aspects of both programs. First, bimD+ function must be executed during S through M. Second, in bimD6 germlings, mitotic nuclear divisions and overall cellular program occur more rapidly than in wild type. Thus, BIMD, an abundant chromosomal protein, is a negative regulator of normal cell cycle progression. Third, bimD6 reduces the level of mitotic interhomolog recombination but does not alter the ratio between crossover and noncrossover outcomes. Moreover, bimD6 is normal for intrachromosomal recombination. Therefore, BIMD is probably not involved in the enzymology of recombinational repair per se. Finally, during meiosis, staining of the Sordaria ortholog Spo76p delineates robust chromosomal axes, whereas BIMD stains all chromatin. SPO76 and bimD are functional homologs with respect to their roles in mitotic chromosome metabolism but not in meiosis. We propose that BIMD exerts its diverse influences on cell cycle progression as well as chromosome morphogenesis and recombination by modulating chromosome structure.

A growth-constrained environment drives tumor progression in vivo

We recently have shown that selective growth of transplanted normal hepatocytes can be achieved in a setting of cell cycle block of endogenous parenchymal cells. Thus, massive proliferation of donor-derived normal hepatocytes was observed in the liver of rats previously given retrorsine (RS), a naturally occurring alkaloid that blocks proliferation of resident liver cells. In the present study, the fate of nodular hepatocytes transplanted into RS-treated or normal syngeneic recipients was followed. The dipeptidyl peptidase type IV-deficient (DPPIV−) rat model for hepatocyte transplantation was used to distinguish donor-derived cells from recipient cells. Hepatocyte nodules were chemically induced in Fischer 344, DPPIV+ rats; livers were then perfused and larger (>5 mm) nodules were separated from surrounding tissue. Cells isolated from either tissue were then injected into normal or RS-treated DPPIV− recipients. One month after transplantation, grossly visible nodules (2–3 mm) were seen in RS-treated recipients transplanted with nodular cells. They grew rapidly, occupying 80–90% of the host liver at 2 months, and progressed to hepatocellular carcinoma within 4 months. By contrast, no liver nodules developed within 6 months when nodular hepatocytes were injected into the liver of recipients not exposed to RS, although small clusters of donor-derived cells were present in these animals. Taken together, these results directly point to a fundamental role played by the host environment in modulating the growth and the progression rate of altered cells during carcinogenesis. In particular, they indicate that conditions associated with growth constraint of the host tissue can drive tumor progression in vivo.

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.

BCL-2-deficient T cells demonstrate accelerated cell cycle progression and increased apoptosis following activation. Increasing the levels of BCL-2 retarded the G0-->S transition, sustained the levels of cyclin-dependent kinase inhibitor p27Kip1, and repressed postactivation death. Proximal signal transduction events and immediate early gene transcription were unaffected. However, the transcription and synthesis of interleukin 2 and other delayed early cytokines were markedly attenuated by BCL-2. In contrast, a cysteine protease inhibitor that also blocks apoptosis had no substantial affect upon cytokine production. InterleUkin 2 expression requires several transcription factors of which nuclear translocation of NFAT (nuclear factor of activated T cells) and NFAT-mediated transactivation were impaired by BCL-2. Thus, select genetic aberrations in the apoptotic pathway reveal a cell autonomous coregulation of activation.

Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target.

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lacI, making the cII assay an attractive alternative for mutant phage recovery for any lambda-based mouse mutagenesis assay system. The frequencies of lambda cII- mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C-->A/T transitions, a large fraction of which occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.

Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME.

Stress protein GRP78/BiP is highly induced in progressively growing tumors and has recently been shown to exert a protective role against lysis by cytotoxic T cells and tumor necrosis factor in vitro. This raises the question whether the in vitro observed protective function of GRP78/BiP translates into the in vivo situation in which tumors grow progressively, killing the host. Herein we report that molecular inhibition of GRP78/BiP induction in the fibrosarcoma B/C10ME, while not affecting in vitro cell proliferation, causes a dramatic increase in apoptotic cell death upon Ca2+ depletion of the endoplasmic reticulum. When B/C10ME cells incapable of inducing GRP78/BiP are injected into mice, tumors are initially formed that, however, regress presumably due to a cytotoxic T-cell response demonstrable by a strong in vitro response to the tumor with spleen cells of regressor mice. Since sensitivity to apoptosis is key to tumor rejection, these results may point to new approaches to the therapy of cancer via regulation of stress protein GRP78/BiP.

1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease believed to be a model for the human disease multiple sclerosis (MS). Induced by immunizing B10.PL mice with myelin basic protein (MBP), EAE was completely prevented by the administration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. 1,25-(OH)2D3 could also prevent the progression of EAE when administered at the appearance of the first disability symptoms. Withdrawal of 1,25-(OH)2D3 resulted in a resumption of the progression of EAE. Thus, the block by 1,25-(OH)2D3 is reversible. A deficiency of vitamin D resulted in an increased susceptibility to EAE. Thus, 1,25-(OH)2D3 or its analogs are potentially important for treatment of MS.

17p (p53) allelic losses, 4N (G2/tetraploid) populations, and progression to aneuploidy in Barrett's esophagus.

Increased 4N (G2/tetraploid) cell populations have been postulated to be genetically unstable intermediates in the progression to many cancers, but the mechanism by which they develop and their relationship to instability have been difficult to investigate in humans in vivo. Barrett's esophagus is an excellent model system in which to investigate the order in which genetic and cell cycle abnormalities develop relative to each other during human neoplastic progression. Neoplastic progression in Barrett's esophagus is characterized by inactivation of the p53 gene, the development of increased 4N (G2/tetraploid) cell fractions, and the appearance of aneuploid cell populations. We investigated the hypothesis that patients whose biopsies have increased 4N (G2/tetraploid) cell fractions are predisposed to progression to aneuploidy and determined the relationship between inactivation of p53 and the development of 4N abnormalities in Barrett's epithelium. Our results indicate that increased 4N (G2/tetraploid) populations predict progression to aneuploidy and that the development of 4N abnormalities is interdependent with inactivation of the p53 gene in Barrett's esophagus in vivo.

Pattern of gp120 sequence divergence linked to a lack of clinical progression in human immunodeficiency virus type 1 infection.

Differential rates of AIDS development and/or T4 lymphocyte depletion in HIV-1-infected individuals remain unexplained. The hypothesis that qualitative differences in selection pressure in vivo may account for different rates of disease progression was addressed in nine eligible study participants from a cohort of 315 homosexual men who have been followed since 1985. Disproportionately fewer changes in variable regions and more in C3 of gp12O were found to be significantly associated with slower disease progression. Our finding provides the first example to demonstrate that differential selection pressure related to the emergence of HIV-1 variants is associated with long term nonprogression. Candidate vaccines that elicit strong selection pressure on C3 of gp120 are likely to provide better protection than those targeting variable regions.