Analysis of telomeric repeats and telomerase activity in human colon carcinoma cells with gene amplification

COLO320DM and COLO320HSR are cell lines derived from a human malignant neuroendocrine colon carcinoma. Both lines have a 30–40-fold amplification of a large DNA domain containing the MYC oncogene. By using fluorescence in situ hybridization techniques with a MYC probe, we could demonstrate that MYC amplicons are contained in a large marker chromosome in COLO320HSR cells, in double minutes (dmin) of COLO320DM cells, and in the interstitial regions of 3–4 additional chromosomes in both cell lines. Amplicons in homogeneous staining regions (HSRs) comprise normal MYC genes, while dmin chromosomes contain PVT/MYC chimeras. Although both cell lines showed similar levels of telomerase activity, the telomere length and telomere distribution in chromosomal termini were considerably lower in COLO320DM than in COLO320HSR cells. This indicates that the average telomere length in cancer cells is regulated no only by the rates of telomerase activity but also by some other non-enzymatic mechanisms.     

DNA discontinuities in the domain of amplified human MYC oncogenes

COLO320DM and COLO320HSR are cell lines derived from a human colon carcinoma. Both lines have an amplification of the MYC oncogene: COLO320DM in the form of double minute chromosomes, COLO320HSR as a large marker chromosome with homogeneously staining regions. We have used pulsed field gel electrophoresis (PFGE) to analyze undigested DNA from both COLO320 cell lines. Upon blotting and hybridization with a MYC probe, the autoradiograms showed the existence of discontinuities in the amplified DNA domains. Additional evidence indicating a preferential concentration of DNA breaks in the region containing the MYC amplicons was obtained with the alkaline unwinding technique. We propose that amplicons are connected by hairpin formation or Hoogsteen pairing between free DNA ends.     

靶向MDR1基因的RNAi稳定逆转结肠癌细胞的多药耐药性

目的： 探讨靶向多药耐药蛋白-1 (MDR1)基因的RNA干扰（RNAi）对结肠癌细胞MDR1/P-gp依赖的多药耐药性的稳定逆转作用。方法：分别构建含编码#4029 MDR1 siRNA和#4123 MDR1 siRNA的质粒载体,并转染COLO 320DM结肠癌多药耐药细胞,采用G418筛选克隆细胞,经实时定量RT-PCR及Western blotting鉴定阳性克隆细胞。MTT法检测细胞活力并计算各抗肿瘤药的IC50。流式细胞术检测细胞周期并计算PI/AI值。流式细胞仪测定细胞内adriamycin药物累积浓度。结果：阳性克隆细胞（clone #4029和clone #4123）的MDR1 mRNA和P-gp的表达均被抑制。COLO 320DM结肠癌亲本细胞adriamycin及vincristine的IC50分别为9.616 μmol/L和0.358 μmol/L,而clone #4029的IC50分别降至1.094 μmol/L和0.023 μmol/L（P<0.01）,clone #4123的IC50分别降至0.780 μmol/L和0.035 μmol/L（P<0.01）。COLO 320DM结肠癌亲本细胞用adriamycin及vincristine处理后其PI/AI值分别为5.68及9.59,而clone #4029的PI/AI值分别降至2.74及3.59（P<0.01）,clone #4123的PI/AI值分别降至2.75及3.24（P<0.01）。COLO 320DM结肠癌亲本细胞用10 μmol/L adriamycin处理后细胞内adriamycin累积浓度为27.92,而clone #4029及clone #4123细胞内adriamycin累积浓度分别增加至187.24和215.57（P<0.01）。结论：稳定转染含编码MDR1 siRNA的质粒载体能稳定逆转结肠癌细胞MDR1/P-gp依赖的多药耐药性。     

Cloning of a non-c-myc DNA fragment from the double minutes of a human colon carcinoid cell line

The cell line COLO 320 DM, derived from an untreated human colon carcinoid tumor, was subcloned to obtain a population (Cl 11) with an average of 37 double minutes (DM) per cell. Fractionation of the chromosomes by differential centrifugation yielded a fraction enriched in DM. DNA isolated from the DM-enriched fraction was inserted into the Pst I site of pBR322. One clone, p446, representative of a number of similar clones, contained a region complementary to genomic unique sequences (region p446U). Southern blot analysis using COLO 320 DNA, and DNA from two other cell lines derived from the same biopsy, COLO 320 HSR and COLO 321 HSR, demonstrated amplification and rearrangement of sequences complementary to p446U when compared with 28 different tumor and normal cell lines, some of which contained DM or homogeneously staining regions (HSR). COLO 320 DM Cl 11 had approximately 110 copies per cell of the p446U sequence, or three copies per DM. COLO 320 HSR, which contained one HSR, had 35 copies per cell, while COLO 321 HSR, which contained two HSR, had 700 copies. In addition, p446U did not hybridize with insert sequences of recombinant plasmid pHM(E + H), which includes the human c-myc coding region, 3 kb of upstream flanking sequences and 0.5 kb of downstream flanking sequences, or with an exon 3 probe, pMYC RI-CLA. Amplification of p446U was also not seen in cell lines containing amplified c-myc or N-myc genes. These results indicate that more than one sequence may be amplified in DM or HSR containing tumor cells, but that they need not be amplified together in other tumors.     

Anti-inflammatory effects of Chinese medicinal herbs on cerebral ischemia

Background

Since oxidative stress has been implicated in a neurodegenerative disease such as Alzheimer's disease (AD), natural antioxidants are promising candidates of chemopreventive agents. This study examines antioxidant and neuronal cell protective effects of various fractions of the methanolic extract of Erigeron annuus leaf and identifies active compounds of the extract.

Methods

Antioxidant activities of the fractions from Erigeron annuus leaf were examined with [2,2-azino-bis(3-ethylbenz thiazoline-6-sulfonic acid diammonium salt)] (ABTS) and ferric reducing antioxidant power (FRAP) assays. Neuroprotective effect of caffeic acid under oxidative stress induced by H₂O₂ was investigated with [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) and lactate dehydrogenase (LDH) assays.

Results

This study demonstrated that butanol fraction had the highest antioxidant activity among all solvent fractions from methanolic extract E. annuus leaf. Butanol fraction had the highest total phenolic contents (396.49 mg of GAE/g). Caffeic acid, an isolated active compound from butanol fraction, showed dose-dependent in vitro antioxidant activity. Moreover, neuronal cell protection against oxidative stress induced cytotoxicity was also demonstrated.

Conclusion

Erigeron annuus leaf extracts containing caffeic acid as an active compound have antioxidative and neuroprotective effects on neuronal cells.     

Difference in the expression of three tight junction proteins, barmotin, occludin, and ZO-1, in phenotypically different human colon cancer cell lines

Epithelioid disorganization is a hallmark of gastrointestinal cancers and is believed to be associated with malignant phenotypes such as invasiveness and the potentiality for metastasis. Although tight junctions (TJs) are known to be crucial for the maintenance of polarized organization of the gastrointestinal epithelium, changes in the TJ proteins in human cancers have not yet been fully elucidated. In this report, we investigated the expression and localization of three TJ proteins-barmotin (7H6 antigen), occludin, and ZO-1-in three phenotypically different human colon cancer cell lines exhibiting differnt grades of epithelioid organization. All three proteins were localized at the most apical part of the cell border corresponding to the site of TJs in T₈₄ cells, in which epithelioid organization was well preserved. In contrast, in COLO320DM cells, which showed no epithelioid phenotypes, occludin was not detectable at either the protein or mRNA level, although barmotin and ZO-1 were present in the cytoplasm. In the third cell line, DLD-1, which showed an epithelioid phenotype intermediate between T₈₄ and COLO320DM, aberrant expression of occludin was found in the basolateral cell membrane. On the other hand, barmotin was present in the cytoplasm, whereas ZO-1 was localized at the cell border. These observations showed that changes in the expression of TJ proteins occur in close correlation with epithelioid disorganization in human colon cancers.     

Analysis of surface properties of human cancer cells using derivatized beads

Standard histochemical analysis of cells and tissues generally involves procedures that utilize a relatively small number of probes such as dyes, and generally requires hours or days to process. Our laboratory has developed a novel method for histochemical surveys of cell surface properties that utilizes a large number of probes (derivatized agarose beads) and takes seconds or minutes to accomplish. In this study, 4 human cell lines (CCL-255 (LS123) human colon cancer cells that are non-tumorigenic in nude mice; CRL-1459 (CCD-18CO) human colon endothelial cells that are non-malignant; CCL-220 (COLO 320DM) human colon cancer cells that are tumorigenic in nude mice; and HTB-171 (NCI H446) human lung carcinoma cells) were tested for their ability to bind to agarose beads derivatized with 51 different molecules. There were statistically significant differences in binding of the 4 cell types to all of the 51 types of beads, but 15 types of beads showed dramatic differences in binding to one or more of the 4 cell types. For example, only HTB-171 (NCI H446) bound to p-aminophenyl-beta-D-glucopyranoside-derivatized beads and only CCL-220 (COLO 320DM) bound to L-tyrosine-derivatized beads. The specificity of cell-bead binding was examined by performing assays in the presence or absence of exogenously added compounds in hapten-type of inhibition experiments. This assay, that utilizes large numbers of novel probes, may help in the development of new libraries of surface properties of specific cell types, with differing degrees of malignancy, that at this time could not be developed by using other available technologies.     

Antihyperglycaemic and antioxidant potentials of Cussonia arborea in alloxan-induced diabetic rats

This study was to evaluate the anti-hyperglycaemic potential of the methanolic stem bark extract of Cussonia arborea in alloxan-induced diabetic rat model. C. arborea extract was well tolerated by the rats at the highest dose of 3,200 mg/kg. The extract at the dose of 500 mg/kg body weight significantly (p?<?0.05) reduced the blood glucose levels of diabetic rats from 16.9?±?4.7 to 5.1?±?1.8 (72.4?±?2.9 % reduction) 6 h post-extract administration. Upon screening of the four fractions obtained after chromatographic techniques, fraction 2 showed the highest anti-hyperglycaemic activity (75.2?±?2.1 % reduction). Further phytochemical studies on fraction 2 revealed that it contains mainly saponin. Fraction 2 showed concentration-dependent antioxidant scavenging activity in 1,1-diphenyl-2-picrynyl phenyl hydrazyl photometric assay. The optimum percentage antioxidant activity was 55 % at the concentration of 400 μg/ml. Ferric reducing antioxidant power (FRAP) value (1.6 μm) of the fraction at 400 μg/ml was statistically comparable to that of 2 μm ascobic acid (reference standard).). It was concluded that the anti-hyperglycaemic property of the extract is attributable to fraction 2. The anti-hyperglycaemic activities of C. arborea could also be attributed to its antioxidant potentials.     

Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis

OBJECTIVES:

The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2.

INTRODUCTION:

The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti‐cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail.

METHODS:

HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0‐4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis.

RESULTS:

Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC₅₀ of 1.6 mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro‐ apoptotic proteins P53, Bax and caspase‐3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti‐apoptotic protein Bcl‐2.

CONCLUSIONS:

Chlorella vulgaris may have anti‐cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase‐3 proteins and through a reduction of Bcl‐2 protein, which subsequently lead to increased DNA damage and apoptosis.     

Selective inhibition of cancer cell invasion by a geranylgeranyltransferase-I inhibitor

A number of small GTPases are involved in cancer cell proliferation, migration and invasion. They need to be prenylated for full biological functions. We have recently reported that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, which block the biosynthesis of farnesylpyrophosphate and geranylgeranylpyrophosphate, inhibit in vitro invasion of human pancreatic cancer cells. In the present study, we examined the effects of two selective inhibitors of prenylation, a farnesyltransferase inhibitor (FTI-277) and a geranylgeranyltransferase type I inhibitor (GGTI-298), on in vitro invasion of cancer cells in a modified Boyden chamber assay. The invasion of COLO 320DM human colon cancer cells was inhibited potently by HMG-CoA reductase inhibitor lovastatin and GGTI-298 but weakly by FTI-277. The treatment of cancer cells with GGTI-298 markedly caused RhoA to decrease in the membrane fraction and accumulate in the cytosolic fraction, whereas it had almost no effect on the translocation of Ras. FTI-277 markedly inhibited membrane localization of Ras, but its inhibitory effect on cancer cell invasion occurred only at doses that affected membrane localization of RhoA. FTI-277 and GGTI-298 decreased the growth potential of COLO 320DM cells, but the inhibitory effect of GGTI-298 was rather selective toward invasion in association with changes in cell morphology and RhoA localization. These results suggest that geranylgeranylation of RhoA by geranylgeranyltransferase type I is critical for cancer cell invasion, and inhibition of geranylgeranyltransferase type I activity should offer a novel approach to the treatment of invasion and metastasis of cancer cells resistant to farnesyltransferase inhibitors. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro antioxidant and cytotoxicity activities of selected indigenous South African medicinal plants

BackgroundMedicinal plants are regarded as a large source of phytochemicals that may have anticancer properties. This could lead to the development of innovative drugs or alternative therapy against cancer.ObjectiveThis study was designed to determine the antioxidant and cytotoxicity effect of 5 selected indigenous South African medicinal plants namely; Bulbine frutescens, Bulbine natalensis, Chlorophytum comosum, Kniphofia uvaria, and Tulbaghia violacea.MethodPhytochemical extracts namely; methanol, 50%, 100% ethanol, and water extracts were prepared from the root and shoot of the plants. The antioxidant effect of methanol extracts of the plant materials was performed using a DPPH assay. A preliminary cytotoxicity screening of the phytochemical extracts in the human colon (Caco-2), cervical (HeLa), and hepatocellular (HepG2) cell lines were determined followed by the half-maximal inhibitory concentration (IC50) using MTT assay.ResultThe methanol root extract of B. natalensis and B. frutescens (33.20% and 26.33% respectively) and shoot extract of K. uvaria (17.10%) showed the highest antioxidant. Out of the 5 plants, only 100% ethanol extract of C. comosum, K. uvaria, and T. violacea caused more than 80% cytotoxicity in HepG2 and Caco-2 cell lines. The shoot of B. frutescens (10.43 µg/ml), K. uvaria (23.0 µg/ml), and root of C. comosum (23.77 µg/ml) were the most active with the highest cytotoxicity.ConclusionC. comosum, K. uvaria, and T. violacea possess significant cytotoxicity that is promising in developing alternative drugs against colon and liver cancers. Our results provided new pieces of evidence for antioxidant and cytotoxic activities of these plants which could be useful for developing new anticancer therapies.     

Hesperidin alleviates oxidative stress and downregulates the expressions of proliferative and inflammatory markers in azoxymethane-induced experimental colon carcinogenesis in mice

Objective

Colon cancer is a common malignant neoplasm causing huge morbidity and mortality worldwide. Current therapeutic interventions are unsatisfying, which necessitates novel chemopreventive strategies. The present study was intended to elucidate the chemopreventive efficacy of hesperidin against azoxymethane (AOM)-induced mouse colon carcinogenesis.

Materials and methods

Swiss albino mice were subjected to intraperitoneal injections of AOM once a week for 3 consecutive weeks. Hesperidin treatments were provided in the initiation or post-initiation phases. The number and multiplicity of aberrant crypt foci (ACF), tumor incidence and antioxidant status were determined. Histopathological analyses, proliferating cell nuclear antigen (PCNA) index and modulations in the expression of inflammatory markers such as nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were studied.

Results

Hesperidin treatments significantly inhibited the number and multiplicities of AOM-induced ACF and tumor incidence. Hesperidin reduced oxidative stress parameters and enhanced antioxidant status. A marked decrease in the PCNA index was evident on hesperidin administration. Hesperidin treatments caused a prominent downregulation of NF-κB and its target molecules iNOS and COX-2, thereby combating inflammation.

Conclusion

This study proves the chemopreventive efficacy of hesperidin against the deleterious traits of colon carcinogenesis including accelerated proliferation, inflammation and persistent oxidative stress.     

CD40配体对结肠癌细胞株的体外抑制作用

研究免疫共刺激分子CD40在结肠癌细胞株的表达及重组可溶性人CD40配体(rshCD40L)对结肠癌细胞株的体外抑制作用。采用流式细胞仪分别检测4株结肠癌细胞(HCT116、Caco-2、SW48和COLO-320)CD40分子的表达,同时利用流式细胞仪检测γ干扰素(IFN-γ)对结肠癌细胞株CD40表达的影响。MTT法检测rshCD40L对结肠癌细胞的抑制作用,TUNEL法检测rshCD40L对结肠细胞的促凋亡作用,同时检测联合rshCD40L和IFN-γ抗结肠癌作用。结果流式细胞仪检测有3株结肠癌细胞(HCT116、Caco-2和SW48)表达CD40分子,IFN-γ能增强CD40+结肠癌细胞CD40分子的表达。rshCD40L能明显抑制CD40+结肠癌细胞的增殖作用(抑制率分别为HCT116:33.5%±5.5%;Caco-2:21.5%±3.6%;SW48:30.1%±4.2%),而对CD40-结肠癌细胞株(COLO-320)无明显抑制作用(P<0.05)。rshCD40L能诱导CD40+结肠癌细胞的凋亡作用(凋亡细胞百分比为HCT116:25.6%±4.52%;Caco-2:15.71%±2.27%;SW48:18.0%±3.7%),而对CD40-结肠癌细胞株(COLO-320)无诱导凋亡作用(P<0.05)。另外,IFN-γ能明显增强rshCD40L对结肠癌细胞的抑制增殖和促凋亡作用。重组人CD40配体与IFN-γ联合可显著诱导CD40阳性的结肠癌细胞凋亡,抑制其增殖,具有潜在的抗肿瘤作用。     

Hepatoprotective effect of acetonic and methanolic extracts of Heterotheca inuloides against CCl4-induced toxicity in rats

A model of hepatotoxicity by carbon tetrachloride (CCl₄) in rats was used in order to evaluate the protective potential of the acetonic and methanolic extracts of Heterotheca inuloides. Pretreatment with the two H. inuloides extracts attenuated the increase in the activity of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) observed in CCl₄-induced liver injury. The protective effect was confirmed by the analysis of tissue slides stained with hematoxylin-eosin and periodic acid/Schiff’s reagent. Additionally, the two extracts are scavengers to the superoxide radical as was observed by electron paramagnetic resonance. Due to the fact that the methanolic extract resulted in a better protective effect in the previous experiments, it was used to investigate in more detail the mechanism of hepatoprotection. Quercetin, one of the main components of the extract, with known hepatoprotective and antioxidant activity was used as a positive control. Pretreatment of animals with the methanolic extract or quercetin, was associated with the prevention of 4-hydroxynonenal and 3-nitrotyrosine increase in the liver, two markers of oxidative stress. Furthermore, the decrease in the activity of several antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase in CCl₄-induced liver injury was alleviated by the pretreatment with H. inuloides methanolic extract or quercetin. These results suggest that the hepatoprotective capacity of H. inuloides methanolic extract is associated with its antioxidant properties, which would also explain the biomedical properties attributed to this plant.     

In Vitro Analysis of Antioxidant Activities of Oxalis Corniculata Linn. Fractions in Various Solvents

As part of our search for natural antioxidants, this work presents an evaluation of antioxidant activities of methanolic extract of Oxalis corniculata and its sub-fractions in hexane, chloroform, ethyl acetate, n-butanol and water. The total phenolic contents in terms of µg of gallic acid equivalents per mg of dried mass were approximately 21.0, 28.2, 34.5, 162.0, 70.0, and 49.2 in methanolic, hexane, chloroform, ethyl acetate, n-butanolic and aqueous fractions respectively, while the flavonoid contents in these solvents were 362.4, 214.1, 317.1, 177.1, 98.8 and 53.5 respectively in terms of µg of rutin per mg of dried mass. In DPPH assay, the ethyl acetate fraction showed the highest free radical scavenging activity, 24.0% with 1 mg/mL concentration. The second strongest fraction was chloroform (21.5%). The EC₅₀ and TEC₅₀ values of the methanolic extract were 3.63 mg/mL and 23 min respectively. The FRAP values in terms of µg of ascorbic acid equivalents per mg of dried mass for these solvents were 288.0, 1705.3, 437.1, 72.0, 28.0, and 44.0 respectively while total antioxidant activity measured by phosphomolybdate assay in terms of µg of ascorbic acid equivalents per mg of dried mass were 50.0, 117.0, 78.6, 57.8, 3.4 and 8.3 respectively. All the samples showed remarkable ability to inhibit lipid peroxidation exhibiting much better and sustainable peroxidation inhibitory activity than the standard butylated hydroxyanisole.     

DNA amplification and chromosomal translocations are accompanied by chromosomal instability: analysis of seven human colon cancer cell lines by comparative genomic hybridization and spectral karyotyping

Genetic instability in human cancers is classified as chromosomal instability (CIN) or microsatellite instability (MIN). DNA amplification and translocations are observed frequently in various cancers. We used comparative genomic hybridization (CGH) and spectral karyotyping (SKY) to study seven human colon cancer cell lines and investigate the relations among genetic instability, DNA amplification, and chromosomal translocations. DNA amplification was found in five cell lines (COLO320DM, COLO201, WiDr, CoCM-1, and CACO-2), and all were aneuploid. In these five cell lines, segments of chromosomes were translocated to other chromosomes. In contrast, cell lines with MIN, DLD-1, and LoVo did not show DNA amplification. The LoVo cells with MIN were considered near diploid and contained translocations. These findings suggest that DNA amplification and chromosomal translocations are accompanied by CIN.     

Anti-Inflammatory,Cytotoxic and Antioxidant Effects of Methanolic Extracts of Drypetes Sepiaria (Euphorbiaceae)

The aim of this study was to investigate in vitro antioxidant, anti-inflammatory and cytotoxic activities of the petroleum ether, ethyl acetate, methanol and aqueous extracts obtained from leaves of Drypetes sepiaria (Euphorbiaceae). Total phenolic and flavonoid contents of these crude extracts were determined as gallic acid and quercetin equivalents, respectively. In in vitro antioxidant method, methanol extract exhibited higher free radical scavenging activity compared to standard compound, ascorbic acid with IC₅₀ of 95.43µg/ml (DPPH) and 67.05µg/ml (ABTS). Methanol extract was able to inhibit inflammation by in vitro about 85–90% (HRBC stabilization method) and in vivo about 40–45% (Paw oedema method) anti-inflammatory assays compared to standard produced 50.04% at 6h period. In cytotoxicity assay (MTT assay) methanolic extract exhibited IC₅₀ of 10µg/ml. In apoptosis (flow cytometric assay), the control group showed normal caspase 3 activity in the SiHa cells which was 0.24%, and increased up to 40% after treatment.     

Proximity of thyroglobulin and c-myc genes on human chromosome 8

The human thyroglobulin structural gene (TG) was mapped to the long arm of chromosome 8 by blot hydridization of a TG cDNA probe to DNA from 21 human × mouse somatic cell hybrids containing overlapping subsets of human chromosomes. In situ hybridization of the TG probe to metaphase chromosomes from a karyotypically normal human lymphoblastoid cell line, JS, localized the TG gene to within the region 8q23 q24.3. Thus, the TG and c-myc genes map to the same chromosome band in normal human cells. In a human colon carcinoma cell line (COLO 320 DM) which contains amplified c-myc, the TG gene is not amplified and hence it lies outside the amplification domain.     

Apoptosis Induction of Centella Asiatica on Human Breast Cancer Cells

The present study evaluated the ability of methanolic extract of Centella asiatica (Linn) Urban (Umbelliferae) to induce apoptosis in different cancer cell lines. MCF-7 cells emerged as the most sensitive cell line for in vitro growth inhibitory activity. C. asiatica extract induced apoptosis in MCF-7 cells as indicated by nuclear condensation, increased annexin staining, loss of mitochondrial membrane potential and induction of DNA breaks identified by TUNEL reactivity. It is possible that the use of C. asiatica extract as a component in herbal medicines could be justifiable.     

Recombination between separate MYC amplification structures in COLO320 cells

Cytogenetically visible gene amplification structures can consist of arrays of amplicons presumably formed by secondary “rearrangements” following amplicon formation. The structural evolution of gene amplification sites in tumor cells suggests that complex secondary structures may have some selective advantage in the tumor cell environment. Although secondary amplicon rearrangements are a hallmark of the gene amplification process, little is known about the mechanics of this process. COLO320 neuroendocrine tumor cells carry two different types of amplified MYC oncogene sequences, one type with an intact MYC gene and the other with a rearranged “chimeric” MYC gene. We have studied various clonal subpopulations of COLO320 cells and identified regions within and downstream of the MYC locus that are unique to each amplicon type. Using double-label fluorescence in situ hybridization with DNA probes unique to each amplicon type, we have observed that both chromosomal and extrachromosomal MYC amplicon arrays in COLO320 cells frequently consist of heterogeneous mixtures of each MYC amplicon type. Our results suggest that the two MYC amplicon types of COLO320 cells were formed simultaneously but independently, and that double minute chromosomes observed in COLO320 cells were formed by intermolecular homologous recombination secondary to amplicon formation. © 1993 Wiley-Liss, Inc.