Abnormal Human Cells Research Articles

Nuances of the Papanicolaou stain.

The impressive list of achievements of Dr. G. N. Papanicolaou and his tedious journey from normal to abnormal human cell includes the importance of wet fixation of cells and the development of the unique polychromatic Pap stain. The 5-dye Pap stain method evolved through 2 salient phases. The first being the development of wet fixation using alcohol-ether to enhance cellular transparency and the second phase saw the introduction of various cytoplasmic counterstaining methods using orange G and EA (light green, Bismarck brown, eosin) and phosphotungstic acid, facilitating the distinction of cell types. The specific characteristics of the staining method is, the cellular transparency combined with crisp nuclear staining, achieved through tailored cellular fixation and cytoplasmic staining using variable dye and pH combinations. With little modifications if any the Pap stain continues to be applied uniformly globally. However, institutional supply of dyes and chemicals from different companies make minor modifications, that remain consistent, an essential part of the staining protocol. This chapter describes the preparation and principles of various components of the stain that are being currently used in our department.

WDR90 is a centriolar microtubule wall protein important for centriole architecture integrity.

Centrioles are characterized by a nine-fold arrangement of microtubule triplets held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in human cells. Altogether, this work highlights that WDR90 is an evolutionary conserved molecular player participating in centriole architecture integrity.

A comparative study of the aneugenic and polyploidy-inducing effects of fisetin and two model Aurora kinase inhibitors

Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements.

Brevilin A, a Novel Natural Product, Inhibits Janus Kinase Activity and Blocks STAT3 Signaling in Cancer Cells

Signal abnormalities in human cells usually cause unexpected consequences for individual health. We focus on these kinds of events involved in JAK-STAT signal pathways, especially the ones triggered by aberrant activated STAT3, an oncoprotein which participates in essential processes of cell survival, growth and proliferation in many types of tumors, as well as immune diseases. By establishing a STAT3 signal based high-throughput drug screening system in human lung cancer A549 cells, we have screened a library from natural products which contained purified compounds from medicinal herbs. One compound, named Brevilin A, exhibited both strong STAT3 signal inhibition and STAT3 signal dependent cell growth inhibition. Further investigations revealed that Brevilin A not only inhibits STAT3 signaling but also STAT1 signaling for cytokines induced phosphorylation of STAT3 and STAT1 as well as the expression of their target genes. In addition, we found Brevilin A could attenuate the JAKs activity by blocking the JAKs tyrosine kinase domain JH1. The levels of cytokine induced phosphorylation of STATs and other substrates were dramatically reduced by treatment of Brevilin A. The roles of Brevilin A targeting on JAKs activity indicate that Brevilin A may not only be used as a STAT3 inhibitor but also a compound blocking other JAK-STAT hyperactivation. Thus, these findings provided a strong impetus for the development of selective JAK-STAT inhibitors and therapeutic drugs in order to improve survival of patients with hyperactivated JAKs and STATs.

Degradation of the Human Mitotic Checkpoint Kinase Mps1 Is Cell Cycle-regulated by APC-cCdc20 and APC-cCdh1 Ubiquitin Ligases*

Mps1 is a dual specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into the G(1) phase. Although evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the anaphase-promoting complex (APC)-c(Cdc20) complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G(1) phase. First, we showed that treatment of HEK 293T cells with the proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell cycle-dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G(1) phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G(1) phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC-c(Cdc20) and APC-c(Cdh1) ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.

Vegetable cells in Papanicolaou-stained cervical smears

Vegetable cells are unusual findings in Papanicolaou-stained cervical smears; these structures could be wrongly mistaken for abnormal human cells, worm eggs, or spores by a cytologist encountering the possibility of meeting those elements in cytological analysis. Five cervicovaginal smears showing similar vegetable cells have been detected over a 3-yr period (2002-2004) in the course of a population screening program for cancer of the uterine cervix in Modena (Italy) involving 32,500 women. According to the clinical histories of the patients, the vaginal pharmaceutical drugs or appliances used were of different types: vaginal lavages, pessaries, and vaginal creams. Following a careful investigation, the only substance that can lead to vegetal elements has been identified as polysaccharide galactomannan, which is one of the excipient present in the drugs used. The authors have identified the origin of these contaminants and the means of pollution, using cytological and pharmaceutical investigation.

Immunological lesions in human uracil DNA glycosylase: association with Bloom syndrome.

Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome.

Insulin stimulation of aminoisobutyric acid transport in human skin fibroblasts is mediated through both insulin and type I insulin-like growth factor receptors.

A monoclonal antibody to the human type I insulin-like growth factor (IGF-I) receptor (alpha IR-3) was used to distinguish actions of insulin and IGF-I that are mediated through insulin as opposed to IGF-I receptors on human skin fibroblasts. Both insulin and IGF-I stimulate uptake of the nonmetabolized alanine analog alpha-aminoisobutyric acid (AIB) in these cells. alpha IR-3 inhibited AIB uptake stimulated by both of these hormones in a dose-dependent manner. However, the pattern of hormone action in the presence of alpha IR-3 differed for the two hormones. In the case of IGF-I, alpha IR-3 potently inhibited AIB uptake at low hormone concentrations, but this inhibition was overcome by high hormone concentrations, consistent with impairment of IGF-I action through the IGF-I receptor. In the case of insulin, the action of low concentrations (i.e. 10 ng/ml) was not inhibited, but that of higher insulin concentrations was, suggesting a dual receptor mechanism of cell stimulation by insulin. alpha IR-3 will be an important tool in further studies of the biology of the IGF-I receptor in normal and abnormal human cells.

Replication of chromosomal DNA in cultured abnormal human cells.

The replication of chromosomal DNA in a series of abnormal human cell cultures has been studied by means of DNA-fiber autoradiography. In lymphocytes with trisomy 21, in fibroblasts of 45,X;47,XXX;49,XXXXY; and 49,XXXXX chromosomal constitution, and in fibroblasts from a patient with xeroderma pigmentosum (De Sanctis-Cacchione syndrome), the rate of DNA replication does not differ from that in normal cells, varying in a single fork from 0.2 to 1.0 micrometer/min with a mean of about 0.6 micrometer/min. In fibroblasts with trisomy 7 the rate of DNA replication is greater, varying from 0.3 to 1.2 micrometer/min with a mean of about 0.8 micrometer/min. The sizes of replication units in all cells examined are from 80 to 500 micrometer with a mean of about 200-300 micrometer.

Dynamics of changes in abnormal human cells during long-term culture in the stationary phase investigation of cells with trisomy 7

Ontogenetic changes in normal diploid cells and cells with chromosomal aberrations (strain LTsCh-162;47,XY, +7) during long-term culture in the stationary phase were studied. The human fibroblasts in culture were found to possess a type of ontogenetic change specific for each culture, and the dynamics of these changes depended on the density of the cell population. Two morphologically different subpopulations of cells, differing in their degree of heterochromatinization of the cell nucleus (under normal conditions), and three different subpopulations of cells in the aberrant strain (47,XY, +7) were found on investigation of the cell nucleus.

Transformation of human cells in culture by N-methyl-N'-nitro-N-nitrosoguanidine.

THE development of in vitro cell culture systems in which healthy cells can be transformed (converted to malignant cells) by chemicals, and detailed studies of the interaction of chemical carcinogens (or their activated forms) and cells, have aided the study of chemical carcinogenesis. In vitro chemical transformation of various rodent cells has been well established1–3. Many attempts have been made to transform various cultured normal or genetically abnormal human cells with chemical carcinogens without success4,5. We have tried to transform human cells from both normal and abnormal individuals and as has been the experience of others, have failed to observe any changes in such cultures. Therefore, the possibility of using continuous lines of human sarcoma cells for chemical transformation was investigated, since certain human sarcoma cell lines are susceptible to transformation by both DNA and RNA tumour viruses6–8. This communication reports results of experiments showing that human osteosarcoma clonal cells can be transformed in vitro by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a known potent carcinogen9,10 and that these transformed cells produced tumours when injected into NIH nude athymic mice.

Identification of group'E' chromosome abnormalities in human cells.

The specific adenovirus type-12 induced aberration in chromosome No. 17 has been used to identify this autosome in cells from patients with No. 17/18 (E group) chromosome abnormalities. The results show that all the cases examined involved abnormalities of chromosome No. 18.

Early events required for induction of chromosome abnormalities in human cells by herpes simplex virus

Studies on chromosome abnormalities induced in human diploid cells by herpes simplex virus type 2 (HSV-2) were undertaken to determine the period of the viral replication cycle during which these abnormalities occur. When cells pretreated for 16–18 hr with human interferon were inoculated with HSV-2 for 4 hr, chromosome abnormalities did not occur except at low levels found in control cultures. Thus, HSV-2 induced chromosome damage occurs following viral uncoating and requires some translation of the viral genome. Other experiments were performed to determine whether these abnormalities occur prior to synthesis of viral DNA. Cytosine arabinoside (ara-C), which is known to inhibit herpes simplex viral DNA synthesis, was used in these experiments. Although ara-C produced a 99.9% inhibition of HSV-2 multiplication, it did not prevent induction of chromosome damage by HSV-2. These results indicate that HSV-2 induced chromosome damage occurs in the absence of extensive viral DNA replication. In fact, ara-C acted synergistically with HSV-2 in the induction of multiple chromosome breaks. The nature of this synergism is unknown.

The influence of progressive growth on the specific catalase activity of human diploid cell strains. I. Effect of cellular genotype: homozygous strains.

AbstractThe specific catalase activity of human diploid cell strains increases with progressive growth of the culture, and falls again following subculture. Although the increase is small, it is readily demonstrable, and is exponential with time.The response of catalase activity to proggressive growth of the culture was studied in three abnormal human cell lines. A diploid cell strain, developed from a patient homozygous for the gene causing acatalasia I, had no detectable catalase activity throughout the life cycle of the culture. Another diploid cell strain, developed from a patient homozygous for the gene causing acatalasia II, had about 5% normal catalase activity, but the proportionate increase in specific activity as the culture grew was the same as for normal cells. Thus the mutation causing acatalasia II does not change the responsiveness of the cell in terms of catalase activity to progressive growth of the culture. The behavior of a heteroploid line was similar to that of the normal diploid strains, but when the growth of the heteroploid cultures reached a plateau, their population densities were four times higher than those of the diploid strains and they had about twice the specific catalase activity.