Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs

Drugs targeting the mitotic spindle are used extensively during chemotherapy, but surprisingly, little is known about how they kill tumor cells. This is largely because many of the population-based approaches are indirect and lead to vague and confusing interpretations. Here, we use a high-throughput automated time-lapse light microscopy approach to systematically analyze over 10,000 single cells from 15 cell lines in response to three different classes of antimitotic drug. We show that the variation in cell behavior is far greater than previously recognized, with cells within any given line exhibiting multiple fates. We present data supporting a model wherein cell fate is dictated by two competing networks, one involving caspase activation, the other protecting cyclin B1 from degradation.     

5alpha-Reductase as a target enzyme for anti-prostatic drugs in organ culture

Short-term organ culture of rat and human prostatic tissues has been utilized as a means of testing drlgs potentially useful in cancer of the prostate. Optimal conditions, particularly the concentration of T, have been established for organ culture of such tissues and the effects of various drugs on 5alpha-reductase activity (5alpha-RA) have been utilized as a means of ascertaining antiprostatic actions of the various compounds and drugs tested. Thus, it was shown that estracyt, estradiol-17beta, progesterone, and novel steroids and steroid-conjugates have definite effects on 5alpha-RA in this system.     

Differential mitotic responses to microtubule-stabilizing and -destabilizing drugs

Although microtubule interacting agents inhibit spindle dynamics, thereby leading to a block in mitosis, we report that low concentrations of these drugs result in differential mitotic effects. Microtubule-stabilizing agents including Taxol, epothilone B, and discodermolide produce aneuploid populations of A549 cells in the absence of a mitotic block. Such aneuploid populations are diminished in an epothilone B-resistant cell line. In contrast, microtubule-destabilizing agents like colchicine, nocodazole, and vinblastine are unable to initiate aneuploidy. The aneuploid cells result from aberrant mitosis as multipolar spindles are induced by the stabilizing drugs, but not by destabilizing agents. The results suggest that the mechanism underlying aberrant mitosis may not be the same as that responsible for mitotic block, and that the former determines the sensitivity of cells to Taxol-like drugs.     

Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level.     

Impact of viral oncogenesis on responses to anti-cancer drugs and irradiation

The view that chemical or physical oncogenesis and tumor therapy resistance represent different parts of common cellular alterations gained considerable attractiveness, because it explains the inherent unreponsiveness of many tumors. Viruses are potent oncogenes and are causally linked to approximately one-fifth of all human malignancies. Whether viral oncogenesis exerts comparable effects was less clear. Recent progress in experimental research provided ample evidence that viruses affect response of tumor cells toward anti-cancer drugs and irradiation. Resistance to cytostatic drugs and radiation develops by alterations at the drug-target sites (i.e., DNA or specific target proteins), upstream (i.e., detoxification mechanisms), or downstream of them (i.e., programmed cell death). Viruses interfere with specific cellular genes at these three levels. Viral proteins induce the expression and expression of drug resistance genes, that is, MDR1, DHFR, or CAD. Viral interactions with the tumor suppressor genes (p53, pRB) abrogate cell cycle arrests and disturb DNA repair of drug- and radiation-induced DNA lesions. The readiness to commit cellular suicide (apoptosis) is also affected by viral genes. The connection between viral oncogenesis and the response of tumor cells to treatment adds a new dimension to tumor biology and may have important consequences for oncological treatment modalities in the future.     

RNA helicase DDX5 is a p53-independent target of ARF that participates in ribosome biogenesis

The p19ARF tumor suppressor limits ribosome biogenesis and responds to hyperproliferative signals to activate the p53 checkpoint response. Although its activation of p53 has been well characterized, the role of ARF in restraining nucleolar ribosome production is poorly understood. Here we report the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of Arf-deficient mouse cells. Through this approach, we discovered that ARF limited the nucleolar localization of the RNA helicase DDX5, which promotes the synthesis and maturation of rRNA, ultimately increasing ribosome output and proliferation. ARF inhibited the interaction between DDX5 and nucleophosmin (NPM), preventing association of DDX5 with the rDNA promoter and nuclear pre-ribosomes. In addition, Arf-deficient cells transformed by oncogenic RasV12 were addicted to DDX5, because reduction of DDX5 was sufficient to impair RasV12-driven colony formation in soft agar and tumor growth in mice. Taken together, our findings indicate that DDX5 is a key p53-independent target of the ARF tumor suppressor and is a novel non-oncogene participant in ribosome biogenesis.     

Cell growth-dependent variation in the sensitivity of human and mouse tumor cells to complement-mediated killing

Cells removed from asynchronous cultures during lag, log, and stationary phases of growth were found to differ in their sensitivity to antibody/complement-mediated killing. The human lymphoblastoid line, Raji, was relatively more susceptible to killing by human anti-HLA antibody plus rabbit complement during the lag and log phases of growth, while the human lymphoid cell line, PY, was relatively more susceptible to rabbit antilymphocyte serum or human anti-HLA plus rabbit complement during the log and late-log phases of growth. The mouse mastocytoma cell line, P815, was relatively resistant to killing by rabbit anti-P815 plus guinea pig complement during the middle log phase of growth. The variation in sensitivity of the three cell lines was dependent upon the concentration of antibody used to sensitize the cells but not due to differences in antigen expression, antigen density, or net synthesis of DNA, RNA, protein, complex carbohydrate, or lipid-containing macromolecules. These data suggest that the variability in susceptibility of the cells for complement-mediated killing is due to changes in physiological and/or physicochemical properties of the cells which either affect the ability of the cells to repair complement-mediated damage or nullify the activity of cell-bound complement.     

PYM-BAG5和131I-BAC5联合导向治疗鼻咽癌的实验研究

背景与目的:现代的肿瘤治疗提倡综合治疗,肿瘤的导向综合治疗具有研究价值.本实验在研究鼻咽癌放射免疫治疗的基础上,观察平阳霉素(pingyangmycin,PYM)和131 I与鼻咽癌单抗BAC5的偶联物PYM-BAC5和131 I-BAC5对鼻咽癌细胞CNE2的联合抑制效果.方法:用葡聚糖T-40作中间载体偶联PYM和BAC5,用测定抑菌活性和免疫活性的方法鉴定偶联物.用氯胺T法制备131 I-BAC5.设置单独用药组(游离PYM,131 I-mIgG)、单独导向用药组(PYM-BAC5,131 I-BAC5)以及联合导向用药组(PYM-BAC5+131 I-BAC5)共5个实验组,用MTT法测定各药物组的抑制作用.结果:PYM-BAC5和游离PYM对CNE2的半数抑制浓度(IC50)分别为46.57 μg/ml和316.70 μg/ml;131 I-BAC5和131 I-mIgG对CNE2的IC50分别为4.42×105 Bq/ml和＞11.10×105 Bq/ml;联合用药组中PYM-BAC5的IC50为7.01 μg/ml,131 I-BAC5的IC50为0.54×105Bq/ml.结论:(1)导向用药组的抑瘤效果明显高于非导向组;(2)联合导向用药组的抑菌效果明显高于单独导向用药组.     

Cell type specific interleukin‐6 induced responses in tumor keratinocytes and stromal fibroblasts are essential for invasive growth

Interleukin‐6 (IL‐6) is one of the major inflammatory interleukins that has been linked to cancer progression. In our model for human skin squamous cell carcinoma (SCC), IL‐6 expression is strongly upregulated upon progression from benign tumors to highly malignant, metastasizing SCCs. We now demonstrate that IL‐6 promotes malignant and invasive tumor growth in human skin SCCs by inducing cell type specific cytokine profiles in tumor keratinocytes and stromal fibroblasts, activating the latter towards a tumor associated fibroblast (TAF) phenotype. In three‐dimensional organotypic cocultures in vitro invasive growth of IL‐6 overexpressing tumor keratinocytes, is associated with increased expression of matrix metalloproteinase‐2 (MMP‐2), MMP‐14 and tissue inhibitor of metalloproteinases‐2, and clearly depends on IL‐6 activated fibroblasts. IL‐6‐induced secretion of monocyte chemotactic protein‐1 (MCP‐1) in tumor keratinocytes and of hepatocyte growth factor in fibroblasts is crucial for regulating expression and activation of MMP‐2. This functional role of IL‐6 is confirmed in vivo. Here MMP‐14 and MMP‐2 expression occur exclusively in surface transplants of IL‐6 overexpressing keratinocytes and fibroblasts are identified as important source of MMP‐2. Our data indicate that tumor keratinocytes derived IL‐6 activates stromal fibroblasts towards a TAF phenotype, promoting tumor invasion via enhanced expression and activation of MMP‐2.     

Prediction of sensitivity to 5-fluorouracil (5-fu) by metabolic and target enzyme activities in colon cancer

BACKGROUND: 5-Fluorouracil (5-FU) and its derivatives are widely known as some of the most commonly prescribed anticancer drugs, especially for gastrointestinal cancer. Orotate phosphoribosyl transferase (OPRT), dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are initial key enzymes in the 5-FU metabolic pathway. The activities of these enzymes may have the potential to affect the chemosensitivity of 5-FU. PURPOSE: This study was designed to investigate the effect of OPRT, DPD and TS in sensitivity to 5-FU. METHOD: We measured OPRT, DPD and TS activities in 11 colonic cancer tissues. The Collagen Gel Droplet Embedded Culture Drug Sensitivity Test (CD-DST) was used in an in vitro chemosensitivity assay. In these samples, the correlation between sensitivity to 5-FU and enzyme activities was investigated. RESULTS: There were no correlations among OPRT, TS activities and sensitivity to 5-FU. In contrast, there was a significant inverse correlation (r=-0.738) between DPD activity and 5-FU sensitivity. With regression analysis, the coefficient of determination of the activity of the three enzymes versus the sensitivity to 5-FU was 0.61. CONCLUSION: Though measuring OPRT, DPD, TS activities is valuable for prediction of sensitivity to 5-FU, DPD is considered to be the most important predictive factor of 5-FU sensitivity. To improve its accuracy, the finding of a fourth factor such as P-glycoprotein and multidrug resistance-associated proteins (MRP), to be added to OPRT, DPD and TS, is desired.     

Cell mediated and humoral immune responses in oral submucous fibrosis

Cell-mediated and humoral immune responses were assessed in 50 patients with oral submucous fibrosis (OSMF) and the results were compared with those of 50 patients with oral-leukoplakia, 50 oral cancer patients, and 50 normal healthy adult controls. The number of high-affinity rosette-forming cells (HARFC) was found to be significantly decreased in OSMF and oral cancer, whereas the number of total rosette-forming cells (TRFC) remained unaltered. Levels of serum IgA, IgD, and IgE were found to be elevated both in OSMF and oral cancer. Immunologic derangements were found to be more pronounced in oral cancer than in OSMF. The enumeration of HARFC along with the estimation of serum levels of IgA, IgD, and IgE indicates that OSMF can be an intermediary stage in the malignant transformation of a normal cell, and that these parameters may serve as markers for the early detection of oral cancer.     

Apoptosis: A Distinctive Mode of Cell Death that Plays an Opposite Role to Mitosis in Cell Population Kinetics

The term apoptosis has been coined for a morphologically distinctive mode of cell death, which plays an opposite role to mitosis in controlling the size of animal tissues: it is fundamentally different from the well known phenomenon of necrosis that results from irreversible injury to cells by agents such as toxins and ischaemia. Cells affected by apoptosis condense and bud to produce many membrane-bounded fragments in which organelles are well preserved. These are phagocytosed and digested by nearby resident tissue cells without associated inflammation; the viable cells merely close ranks. Extensive deletion of cells may thus take place without disorganisation of overall tissue architecture. Apoptosis occurs continuously in healthy animals. It is enhanced in endocrine-dependent tissues during the reversible shrinkage that follows appropriate hormone withdrawal or administration, and it is also implicated in total and irreversible involution of tissues in normal animals, such as loss of the tadpole tail during metamorphosis and elimination of phylogenetic vestiges during embryonic development. Apoptosis takes place spontaneously and continuously in malignant neoplasms, often grossly retarding their growth, and cytotoxic drug administration and X irradiation enhance apoptosis as well as inhibiting mitosis in neoplasms; tumour regression following therapy with these agents is the result of tipping the balance between the two processes. Hyperthermia enhances apoptosis in foetal tissues and adult seminiferous tubules; its effect on apoptosis in neoplasms needs study. Cell mediated immune attack on cells induces apoptosis, not necrosis; apoptosis may thus sometimes be enhanced in neoplasms by immunological means. The features of apoptosis suggest that it is an active process of cellular self-destruction rather than a form of cell degeneration. This carries the implication that it is genetically programmed. Further understanding of the regulation of the onset of apoptosis at the molecular level may lead to the discovery of improved methods of tumour treatment.     

Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs

The epidermal growth factor receptor (EGFR) is a rational target for cancer therapy because it is commonly expressed at a high level in a variety of solid tumours and it has been implicated in the control of cell survival, proliferation, metastasis and angiogenesis. However, despite evidence to suggest that EGFR expression is associated with a poor prognosis in some tumours (e.g. breast, head and neck carcinomas), the situation is by no means clear-cut. A number of issues are worthy of particular consideration, including how EGFR is measured and whether these assays are sensitive and reproducible, which mechanisms other than increased EGFR expression might cause the EGFR signalling drive to be increased, and the relationship, if any, between EGFR expression and the response to EGFR-targeted agents.