Curcumin induces cell death without oligonucleosomal DNA fragmentation in quiescent and proliferating human CD8+ cells

Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemotherapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective chemopreventive agent. Methods: We treated human quiescent or proliferating CD8+ cells with 50 microM curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell viability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observation, Western blotting and flow cytometry. Results: Curcumin decreased cell viability, activated caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease. However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes characteristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be particularly undesirable.     

H1 variant synthesis in proliferating and quiescent human cells

The synthesis of histone H1 isoprotein species in human cells of several different types and in several different physiological states was studied. Up to five H1 and two H1 degrees isoprotein species could be resolved by two-dimensional electrophoresis. All five H1 isoprotein species were synthesized in exponentially growing cultures of IMR-90 human fibroblasts; in quiescent IMR-90 cells the synthesis of three H1 isoprotein species was greatly decreased while the synthesis of two others was much less affected. When DNA synthesis in exponentially growing cultures of IMR-90 was inhibited, the pattern of H1 isoprotein synthesis became similar to that found in quiescent cultures. Other human cells, isolated from blood, yielded similar results. These results suggest that the pattern of H1 synthesis is the same for cells in non-S phases of the cell cycle and in quiescent cells. Thus for histone H1 in human cells the relationship of the variant synthesis pattern to the growth state and DNA replication is similar to that of the core histone H3 but not that of H2A.     

High efficiency protein transduction of quiescent and proliferating primary hematopoietic cells

Primary hematopoietic cells are relatively refractory to DNA transfection methodologies. This is particularly so when they are quiescent or terminally differentiated and no longer able to divide. However, whole proteins can be introduced into such cells by protein transduction. We have modified the protein transduction domain (PTD) from the HIV-TAT protein used by other investigators. Using green fluorescent protein (GFP) as a reporter, we show that this new sequence allows more efficient transduction of recombinant fusion protein into a variety of hematopoietic cells tested compared with the native HIV TAT domain. This is true for peripheral blood CD34+ cells, dendritic cells, granulocytes, monocytes and lymphocytes all of which are quiescent or terminally differentiated. Furthermore, we were able to transduce myeloblasts from patients with acute myeloid leukemia (AML). In all cell types tested transduction efficiency was almost 100%. Transduction is maximal 15-30 s after addition of PTD or TAT-GFP fusion proteins as tested on quiescent T lymphocytes. This method will allow us to study of the effects of a variety of gene products in cell types that were previously resistant to gene transfection studies.     

Modelling complex populations formed by proliferating,quiescent and quasi-quiescent cells: application to plant root meristems

A proliferating population of cells may be considered complex when its proliferative or growth fraction P is lower than 1 and/or when it is formed by subpopulations with different mean cycle times. The present paper shows that in such complex populations exponential growth is consistent with a steady-state distribution of cells. Obviously, when P=1 then cell distribution is only a function of cell age. An analytical model has been developed to study complex populations including both quiescent fractions formed by cells with unreplicated genome (G(0) cells) and cells with fully duplicated chromosomes (Q(2) cells). The model also considers those quasi-quiescent cells in their last transit through G(1) and S (Q(1) and Q(s) cells) before becoming quiescent. In order to solve the difficulties of a direct analysis of the whole population, its kinetic parameters have been obtained by studying the negative exponential distribution of two subpopulations: one formed by the proliferating cells and another formed by the quasi-quiescent cells. Additionally, the model could be applied when quiescence is initiated at any other cycle phase different from G(1) and G(2), for instance, cells in the process of replicating their DNA or being at any other mitotic phases. The utility of the method was illustrated in populations which constitute the root meristems of both Allium cepa L. and Pisum sativum L. Three facts should be stressed: (1) the method seems to be rather powerful because it can be carried out from different sets of experimentally measured parameters; (2) the rate of division and, therefore, the population doubling time can be easily estimated by this method; and (3) it also allows the determination of the amount of cells that had become quiescent either before they had replicated their DNA (G(0)) or after having completed their replication (Q(2)), as well as those quasi-quiescent cells which are progressing throughout their last pre-replicative and replicative periods (thus Q(1) and Q(s), respectively).     

Differential in vitro growth properties of cells transformed by DNA and RNA tumor viruses

Mammalian cells transformed by DNA and RNA tumor viruses are shown to display consistently different growth properties. All SV40, adenovirus type 7 and polyoma virus (DNA viruses) transformed cells propagated to high densities. The same cells transformed instead by RNA viruses: MSV strain Kirsten (MSV-Ki) or MSV strain Maloney (MSV-M) grew to densities which were consistently lower than DNA virus-transformed cells but greater than that of untransformed cells. The capacity to synthesize DNA at increasing densities also differentiated the RNA and DNA virus-transformed cells. As growing cultures of untransformed cells neared saturation density, the fraction of cells synthesizing DNA was minimal. The RNA virus-transformed cells were also contact-inhibited but at a significantly higher density. In contrast the DNA virus-transformed cells propagated to still greater densities and continued DNA synthesis at a high rate even at very high densities. Therefore the DNA virus-transformed cells truly are not contact inhibited. It is suggested that the capacity to continue DNA synthesis at high densities explains the attainment of much greater densities by DNA virus-transformed cells. There were no clear-cut differences in the ability to form colonies in agar, although a few of the RNA virus-transformed lines could not be propagated in semi-solid medium. These results may be explained as a persistence of the capacity of DNA tumor viruses to stimulate host cell DNA synthesis.     

Cytochemical identification of quiescent and growth-activated tumor cells

Cytochemical determinations of the amount of protein in the cell nucleus together with the amount of DNA give a background for judging the growth activity of the individual cell. A series of human breast carcinomas of different malignancy grades was studied, and it was shown that judged by their types of DNA profiles, the tumors of low malignancy grades were characterized by growth-arrested cells while the majority of cells in highly malignant tumors were intensely growth activated. This indicates that determinations of proteins in the nucleus together with DNA determinations can be used to better judge malignancy grades in individual breast carcinomas, which is of considerable clinical importance.     

Phosphorylation of nuclear and DNA-binding proteins in proliferating and quiescent mammalian cells.

The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins.     

Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta

The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.     

DNA replication in quiescent cell nuclei: regulation by the nuclear envelope and chromatin structure

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.     

DNA tumor viruses and human cancer

There is a strong association between viruses and the development of human malignancies. A group of oncogenic DNA viruses exists in the human population today, members of which serve as infectious agents of cancer worldwide. The group includes the Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, human papillomaviruses and human polyomaviruses. Globally, it is estimated that 20% of all cancers are linked to infectious agents. Studies of DNA viruses have contributed to our current understanding of the key molecular players in the transformation process. Research has also shed light on the molecular mechanisms of tumorigenesis that are employed by these viruses and there are indications that cofactors could be required for viral oncogenicity in some cases.     

Senescent and quiescent cell inhibitors of DNA synthesis : Membrane-associated proteins

Cytoplasts derived from senescent and quiescent human diploid cells inhibit DNA synthesis initiation when fused with cells capable of proliferation. When the cytoplasts were subjected to a variety of conditions (trypsin and cycloheximide treatment and growth on fibronectin), this inhibitory activity was lost, suggesting that the inhibitors involved were proteins associated with the surface membranes of the cells. We have studied the quiescent cell inhibitor in greater detail and determined that surface membrane-enriched preparations isolated from quiescent cells and proteins extracted from these membrane preparations have DNA synthesis-inhibitory activity.     

Myeloid cell transformation by ras-containing murine sarcoma viruses.

BALB and Harvey murine sarcoma viruses contain ras transforming genes capable of altering the proliferation and differentiation of cells within the erythroid and lymphoid lineages (W. D. Hankins and E. M. Scolnick, Cell 26:91-97, 1981; J. H. Pierce and S. A. Aaronson, J. Exp. Med. 156:873-887, 1982; E. M. Scolnick et al., Mol. Cell. Biol. 1:68-74). The present studies demonstrate hematopoietic targets of ras-containing viruses within the myeloid lineage. Diffuse colonies were induced by BALB or Harvey marine sarcoma virus infection of murine bone marrow cells. Generally, these colonies were made up of relatively mature macrophages which exhibited increased self-renewal capacity but eventually underwent terminal differentiation in culture. Cells from one BALB murine sarcoma virus-induced colony displayed phenotypic markers of more immature myelomonocytic cells. This colony, designated BAMC1, readily established as a continuous cell line and was highly malignant in vivo. Exposure of these cells to 12-O-tetradecanoylphorbol-13-acetate led to the induction of a more mature myeloid phenotype, which was associated with decreased growth potential in vitro and in vivo. The effects of the inducing agent were not mediated by an alteration in the level of expression of the ras-coded p21 transforming protein. Our present findings extend the spectrum of targets whose growth is altered by ras-containing retroviruses to cells at several stages of differentiation within each of the major hematopoietic lineages.     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

At a crossroads: human DNA tumor viruses and the host DNA damage response

Human DNA tumor viruses induce host cell proliferation in order to establish the necessary cellular milieu to replicate viral DNA. The consequence of such viral-programmed induction of proliferation coupled with the introduction of foreign replicating DNA structures makes these viruses particularly sensitive to the host DNA damage response machinery. In fact, sensors of DNA damage are often activated and modulated by DNA tumor viruses in both latent and lytic infection. This article focuses on the role of the DNA damage response during the life cycle of human DNA tumor viruses, with a particular emphasis on recent advances in our understanding of the role of the DNA damage response in EBV, Kaposi's sarcoma-associated herpesvirus and human papillomavirus infection.