Thomas Heiden

Molecular mechanisms underlying interferon-α-induced G0/G1 arrest: CKI-mediated regulation of G1 Cdk-complexes and activation of pocket proteins

One prominent effect of IFNs is their cell growth-inhibitory activity. The mechanism behind this inhibition of proliferation is still not fully understood. In this study, the effect of IFN-α treatment on cell cycle progression has been analysed in three lymphoid cell lines, Daudi, U-266 and H9. Examination of the growth-arrested cell populations shows that Daudi cells accumulate in a G0-like state, whereas U-266 cells arrest later in G1. H9 cells are completely resistant to IFN-αs cell growth-inhibitory effects. The G0/G1-phase arrest is preceded by a rapid induction of the cyclin-dependent kinase inhibitors (CKIs), p21 and p15. In parallel, the activities of the G1 Cdks are significantly reduced. In addition to p21/p15 induction, IFN-α regulates the expression of another CKI, p27, presumably by a post-transcriptional mechanism. In the G1 Cdk-complexes, there is first an increased binding of p21 and p15 to their respective kinases. At longer exposure times, when Cdk-bound p15 and p21 decline, p27 starts to accumulate. Furthermore, we found that IFN-α not only suppresses the phosphorylation of pRb, but also alters the phosphorylation and expression of the other pocket proteins p130 and p107. These data suggest that induction of p21/p15 is involved in the primary IFN-α response inhibiting G1 Cdk activity, whereas increased p27 expression is part of a second set of events which keep these Cdks in their inactive form. Moreover, elevated levels of p27 correlated with a dissociation of cyclin E/Cdk2-p130 or p107 complexes to yield cyclin E/Cdk2-p27 complexes. In resistant H9 cells, which possess a homozygous deletion of the p15/p16 genes and lack p21 protein expression, IFN-α causes no detectable changes in p27 expression and, furthermore, no effects are observed on either pocket proteins in this cell line. Taken together, these data suggest that the early decline in G1 Cdk activity, subsequent changes in phosphorylation of pocket proteins, and G1/G0 arrest following IFN-α treatment, is not primarily due to loss of the G1 kinase components, but result from the inhibitory action of CKIs on these complexes.

Expression of glycolytic isoenzymes in activated human peripheral lymphocytes: cell cycle analysis using flow cytometry.

Human peripheral lymphocytes activated with concanavalin A and phorbol myristate ester exhibit an increase in glycolysis on a time-course similar to that for DNA synthesis. Elevated glycolysis is accompanied by increased specific activities of the glycolytic enzymes. Increased enzyme activities are accounted for by the appearance of specific isoenzyme forms (muscle forms) normally expressed in rapidly growing tumor cells or in growth-stimulated cells. In the present study we analyzed the expression of the glycolytic isoenzymes during cell cycle progression of activated human lymphocytes using two-parameter (DNA and protein) flow cytometry. Time-course studies and analysis of subpopulations prepared by elutriation centrifugation showed that the inducible isoenzymes are expressed at low levels or not at all in G0 cells. They are expressed first during the G0 to G1 transition or in early G1. However, expression increases throughout G1, reaching a maximum in S-phase. Thus, induction of glycolytic isoenzymes provides an excellent marker of T-cell activation and progression toward DNA synthesis.

Diagnostic DNA‐flow‐ vs. ‐image‐cytometry in effusion cytology

Aims: To determine the sensitivity and specificity of flow‐ and image‐cytometry for the detection of DNA‐aneuploidy as a marker for malignant cells in effusions. Methods: 200 effusions (80 tumor cell‐positive, 74 negative and 46 cytologically equivocal) were stained with DAPI‐SR for DNA‐flow‐ and with Feulgen‐Pararosaniline for ‐image‐cytometry. They were measured using a PAS‐flow‐cytometer and an AutoCyte‐QUIC‐DNA‐workstation according to the ESACP consensus reports for DNA‐flow‐ and ‐image‐cytometry, respectively [7,23,29,49]. Results: Sensitivity of DNA‐aneuploidy for the identification of malignant cells was 32.1% for DNA‐flow‐ and 75.0% for ‐image‐cytometry, specificity of ‐euploidy in benign cells was 100.0% for both methods. Positive predictive value of DNA‐aneuploidy for the identification of malignant cells was 100.0% for both techniques, negative predictive value of DNA‐euploidy was 48.6% for DNA‐flow‐ and 72.0% for ‐image‐cytometry. Conclusions: Searching for DNA‐aneuploidy as a diagnostic marker for neoplastic cells in serous effusions image‐cytometry revealed superior sensitivity as compared with monoparametric flow cytometry.

Proliferation and apoptosis in the evolution of endemic and acquired immunodeficiency syndrome-related Kaposi's sarcoma

Kaposis sarcoma (KS) is a multifocal lesion that occurs predominantly in the skin, most frequently in people infected with HIV-1, and that evolves through early stages (patch and plaque) to a tumor-like late stage (nodular). Both, endemic African (EKS) and AIDS-associated (AKS) KS expressed human herpesvirus 8 (HHV-8) as shown by PCR. By immunohistochemistry the expression of cellular Bcl-2 and c-myc was confined in early stages of both EKS and AKS to relatively few endothelial cells (EC) whereas in nodular KS most of spindle cells (SC) strongly expressed both genes. CD40 was usually strongly expressed in SC at all KS stages as well as in EC of non-involved tissue whereas CD40L (CD154) was not demonstrable. Fas (CD95) was moderately to weakly expressed by SC whereas p53 and Waf-1 were found in less than 5% of the SC. In both AKS and EKS at nodular stage almost no apoptotic SC were detected. In most AKS and EKS low levels of cell proliferation were seen but AKS showed consistently higher values compared to EKS. All clinical types and stages of KS showed a diploid cellular DNA content by flow cytometric analysis of microselected lesions. Thus, we conclude that KS during evolution represents diploid, probably reactive, cell proliferation, which progressively increases the expression of strong cellular and also viral (HHV-8) antiapoptotic factors.

Human herpesvirus 8/Kaposi sarcoma herpesvirus cell association during evolution of Kaposi sarcoma.

Kaposi sarcoma (KS) is associated with a herpesvirus (HHV-8/KSHV), which expresses a latency-associated nuclear antigen (LANA). The histopathology of KS is characterized by angiogenesis, inflammatory cells, and the development of CD34+ tumor spindle cells (SCs). However, the cellular basis for the recruitment and dissemination of HHV-8 during the development of KS lesions is not clear. Twenty-nine KS biopsies with AIDS (AKS, n = 22) and without HIV infection (endemic KS or EKS, n = 7) were immunostained by a triple antibody method to characterize HHV-8-infected and noninfected (LANA+/−) CD34+ SCs, infiltrating CD3+, CD68+, CD20+, and CD45+ leukocytes as well as proliferating (Ki67+) cells. The CD34+/LANA+ SCs were more frequent in late (nodular) as compared with early (patch/plaque) KS stages. However, in late AKS 36.0% of SCs (median of 11 cases) were CD34+/LANA− compared with 20.7% in early cases (median of 11 cases). Furthermore, both AKS and EKS showed, at all stages, a small (4.1–6.5%) population of LANA+/CD34− cells. Proliferating Ki67+ cells were seen (4.5–11.5%) at all KS stages, and were usually more frequent in early AKS, but no significant difference was observed between nodular AKS and EKS. Most of the proliferating cells in the KS lesions were LANA+/CD34+ but a small fraction was LANA+/CD34−. Lesional CD68+ and CD3+ cells varied between AKS (7.3 and 5.2%, respectively) and EKS (4.9 and 3.1%, respectively) but were not clearly stage related. No LANA+ cells were CD3+, CD20+, or CD45+ and very few (<0.5%) were CD68+. These results indicate that not all CD34+ KS SCs were LANA+, suggesting recruitment of noninfected SCs to the lesions. Cell proliferation in general was much higher in early as compared with the late AKS stages. LANA+ SCs could have a proliferative advantage as suggested by higher frequency of cycling (Ki67+) LANA+ SCs. Few macrophages but no lymphocytes are LANA+.

CGH of microdissected Kaposi's sarcoma lesions reveals recurrent loss of chromosome Y in early and additional chromosomal changes in late tumour stages.

Background:It is still unclear if Kaposis sarcoma (KS) is a monoclonal cell proliferation or a polyclonal, hyperplastic, reactive process. Reports on KS cytogenetics are few and restricted to late stage disease and cell lines. Method:We analysed 27 KS, early and late, AIDS related (AKS) and endemic (EKS) by laser microdissection, global DNA amplification and comparative genomic hybridization (CGH). Result:Loss of Y chromosome was detected in 20/23 male KS, which was the only recurrent chromosomal aberration in all nine male early (patch) KS. Only one patch EKS showed in addition to the Y loss a loss of Xq. Late (nodular) AKS and EKS showed recurrent copy number changes in chromosomes 16, 17, 21, X and Y, as well as other random changes. The loss of chromosome 16, 17 and Y was confirmed by interphase fluorescence in situ hybridization (FISH) on paraffin sections. EKS showed a higher number of chromosomal abnormalities than AKS, indicating that rapid growth of AKS is less dependent on genetic changes than is EKS, possibly because of the immunosuppressed host environment in AKS. Conclusion:Clonal loss of chromosome Y was detected in all early male KS, while additional chromosomal aberrations appeared during development to late KS. This increase in chromosomal abnormalities during tumour growth indicates genetic instability and the selection of survival cell clones establishing late, aggressive sarcoma growth. Our data support the view that KS (in males) develops into a clonal tumour yet initially is a hyperplastic reactive cell proliferation.

Dichotomy between CD1a+ and CD83+ dendritic cells in lymph nodes during SIV infection of macaques.

Abstract:  The prevalence and differentiation of dendritic cells (DC) in lymphoid tissue of simian immunodeficiency virus (SIV)‐infected cynomolgus monkeys was studied during disease progression. Lymph node biopsies were consecutively obtained from clinical rapid and slow progressors until the development of disease consistent with simian acquired immunodeficiency syndrome (sAIDS) occurred. Quantitative evaluation of CD1a+ DC and the expression of DC antigens related to maturation (CD83, DC‐LAMP and S100b) were performed at the single cell level by in situ image analysis. Despite a persistent prevalence of CD1a+ DC in lymphoid tissue during disease progression, there was a subsequent drop of mature CD83+, DC‐LAMP+ and S100b+ DC, correlating with the decline of CD4+ T cells in blood. Thus, disease progression to sAIDS was associated with impaired maturation of DC, and lack of CD83, DC‐LAMP and S100b expression.

Comparison of routine flow cytometric DNA analysis of fresh tissues in two laboratories: Effects of differences in preparation methods and background models of cell cycle calculation

Routine flow cytometric DNA analysis was compared in two laboratories by using matched fresh-frozen breast cancer and soft tissue sarcoma biopsy specimens. Laboratory I applied the Vindelöv preparation method and an exponential background subtraction algorithm in the cell cycle calculation. Laboratory II used the Formalin-protease preparation technique and the sliced-nuclei background model. The results of the ploidy analysis showed good agreement between the two laboratories; however, the results of the cell cycle analysis showed considerable systematic differences between labs. Laboratory I obtained significantly lower values of S-phase fraction and higher values of G2-phase fraction than laboratory II. To explain these discrepancies, the effects of differences in the preparation methods and background subtraction algorithms were studied. The Vindelöv preparation method yielded higher debris and aggregation levels than the Formalin-protease technique and tended to give higher %S and %G2 values. When the two background models were used in the same histograms, the exponential background model tended to give %S values distinctly lower than and %G2 values almost identical to those obtained with the sliced-nuclei algorithm. The sum of these effects accounts for the observed inter-laboratory discrepancies. Different from the sliced-nuclei fit, the exponential background fit often did not accommodate to the original data in the <2c histogram region and resulted in a considerable inter-operator variability of %S calculation in histograms with <5% S. When aggregate correction was added to the sliced-nuclei algorithm, the differences between %S values in histograms from the two laboratories almost disappeared.

Combined Analysis of DNA Ploidy, Proliferation, and Apoptosis in Paraffin-Embedded Cell Material by Flow Cytometry

A flow cytometric assay was developed for correlated measurement of DNA content and apoptotic DNA strand breaks in cell nuclei of formalin-fixed, paraffin-embedded tissues. The assay allows a combined analysis of cell ploidy, proliferation, and apoptosis in sections of fixed paraffin-embedded archival or fresh tissue/cell specimens. It is based on (a) proteolytic release of cell nuclei from deparaffinized and rehydrated 90-μm thick sections of the fixed embedded specimen, (b) the inactivation of the protease, (c) FITC-labeling of DNA strand breaks by the terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP nick end-labeling (TUNEL) reaction, and (d) DNA staining with 4′6-diamidino-2-phenyleindole. The fluorescence was recorded with a double-beam flow cytometer equipped with a mercury arc lamp and an argon ion laser. Cytograms obtained with this assay correlated closely with those produced using nonembedded material from the same specimen. Furthermore, a significant correlation was found between flow cytometric analysis of apoptosis in cell nuclei released from paraffin blocks and conventional evaluation of TUNEL on (corresponding) sections (p < 0.001). Since necrotic cells can stain positively by TUNEL, the possibility to microscopically select nonnecrotic tumor regions for flow cytometric analysis is an important advantage of the assay.

Simian AIDS-Related Lymphoma Growth in Severe Combined Immunodeficiency Mice Is Independent of Karyotypic Abnormalities or Bcl-6 Mutations

Simian AIDS-related lymphomas (sARL) of cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied in relation to growth in severe combined immunodeficient (SCID) mice, karyotype abnormalities, and DNA sequence of the first noncoding region of the Bcl-6 gene. The tumors were diffuse large B cell lymphomas and expressed a simian homolog to Epstein-Barr virus (HVMF-1) in 12 of 13 primary tumors and corresponding cell lines. A tested cell line was tumorigenic in SCID mice. Tumors in the SCID mice showed cell growth features similar to those in the original lymphoma, suggesting that no subpopulation with growth advantage was selected for in the mice. Spectral karyotype analysis of sARL cell lines showed normal cytogenetic features except for a trisomy of monkey chromosome 2 (corresponding to human chromosomes 7 and 21) in two of five sARL lines, which was not recovered in SCID tumors established from the same cell line. Sequence analysis of a Bcl-6 gene fragment showed sequence variations indicative of population polymorphism(s) in 10 of 13 sARLs, and no evidence of Bcl-6 mutations. Thus Bcl-6 mutations in the first noncoding region are irrelevant for sARL development in cynomolgus monkeys and for tumorigenicity of sARL cell lines. We also demonstrate that no cytogenetic alterations are needed for the development of highly aggressive lymphomas in the SIV-immunosuppressed host.