The analysis of malignancy by cell fusion. VIII. Evidence for the intervention of an extra-chromosomal element

Diploid human fibroblasts and lymphocytes were fused with the cells of a malignant mouse melanoma and a range of hybrid clones selected for study. The ability of these clones to produce progressive tumours was assayed in nude mice. Although human chromosomes were preferentially eliminated in all the hybrid clones, the human diploid cells were as effective as mouse diploid cells in suppressing the malignancy of the mouse melanoma cells. The suppression produced by fibroblasts was again more profound than that produced by lymphocytes. Malignancy was also found to be suppressed in a hybrid clone in which a single X was the only human chromosome present; and this clone continued to give a very low take incidence even after the human X had been eliminated by back selection. Hybrids were made between the melanoma cells and diploid human fibroblasts that had been given 100 J kg-1 of gamma radiation before cell fusion. These hybrids contained no recognizable human chromosomes, but their ability to produce progressive tumours was greatly reduced compared to that of the melanoma parent cells. The take incidences given by the crosses between the melanoma cells and the irradiated human fibroblasts were, however, substantially higher than those given by the crosses between the melanoma cells and unirradiated fibroblasts. These findings suggest that the suppression of malignancy involves the activity of some extra-chromosomal element and that this element is radio-sensitive.

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Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2814-2820.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2814-2820

Author(s):

T G Lugo ◽

B Handelin ◽

A M Killary ◽

D E Housman ◽

R E Fournier

Keyword(s):

High Efficiency ◽

Human Fibroblasts ◽

Retroviral Vector ◽

Human Chromosomes ◽

G418 Resistance ◽

Microcell Hybrid ◽

Dominant Selectable Marker ◽

Hybrid Clones ◽

Microcell Fusion

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.

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Imputation-free reconstructions of three-dimensional chromosome architectures in human diploid single-cells using allele-specified contacts

10.1101/2021.10.04.462972 ◽

2021 ◽

Author(s):

Yoshito Hirata ◽

Arisa H. Oda ◽

Chie Motono ◽

Masanori Shiro ◽

Kunihiro Ohta

Keyword(s):

Chromosome Structure ◽

Single Cells ◽

Three Dimensional ◽

Recurrence Plot ◽

Human Chromosomes ◽

Fractal Structures ◽

Homologous Chromosomes ◽

Allele Specific ◽

Human Diploid Cells ◽

Diploid Cells

AbstractThe sparseness of chromosomal contact information and the presence of homologous chromosomes with very similar nucleotide sequences make Hi-C analysis difficult. We propose a new algorithm using allele-specific single-nucleotide variations (SNVs) to reconstruct the three-dimensional (3D) chromosomal architectures from the Hi-C dataset of single diploid cells. Our algorithm has a function to discriminate SNVs specifically found between homologous chromosomes to our “recurrence plot”-based algorithm to estimate the 3D chromosome structure, which does not require imputation for ambiguous segment information. The new algorithm can efficiently reconstruct 3D chromosomal structures in single human diploid cells by employing only Hi-C segment pairs containing allele-specific SNVs. The datasets of the remaining pairs of segments without allele-specific SNVs are used to validate the estimated chromosome structure. This approach was used to reconstruct the 3D structures of human chromosomes in single diploid cells at a 1-Mb resolution. Introducing a subsequent mathematical measure further improved the resolution to 40-kb or 100-kb. The reconstruction data reveals that human chromosomes form chromosomal territories and take fractal structures where the mean dimension is a non-integer value. We also validate our approach by estimating 3D protein/polymer structures.

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Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2814 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2814-2820 ◽

Cited By ~ 50

Author(s):

T G Lugo ◽

B Handelin ◽

A M Killary ◽

D E Housman ◽

R E Fournier

Keyword(s):

High Efficiency ◽

Human Fibroblasts ◽

Retroviral Vector ◽

Human Chromosomes ◽

G418 Resistance ◽

Microcell Hybrid ◽

Dominant Selectable Marker ◽

Hybrid Clones ◽

Microcell Fusion

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MORPHOLOGIC CHANGES ACCOMPANYING SENESCENCE OF CULTURED HUMAN DIPLOID CELLS

Journal of Experimental Medicine ◽

10.1084/jem.131.6.1211 ◽

1970 ◽

Vol 131 (6) ◽

pp. 1211-1222 ◽

Cited By ~ 160

Author(s):

E. Robbins ◽

E. M. Levine ◽

H. Eagle

Keyword(s):

Cancer Cells ◽

Marked Decrease ◽

Human Fibroblasts ◽

Cellular Growth ◽

Culture Initiation ◽

Cell Strains ◽

Human Diploid Cells ◽

Diploid Cells ◽

Glycogen Particles ◽

Morphologic Changes

The lysosomes of serially propagated human fibroblasts gradually transform to residual bodies which increase in number and size, and show progressive degenerative changes. There is an accompanying, and less regular, decrease in the number of cytoplasmic polyribosomes and an increased number of glycogen particles. The onset of these morphologic alterations occurs shortly after culture initiation and precedes any marked decrease in the rate of cellular growth; however, in their extreme form these changes may be related to the ultimate cessation of cellular multiplication ("senescence"). The lysosomal changes were seen only in those cell strains which eventually showed senescence, and were absent or minimal either in cell lines which can be propagated indefinitely ("spontaneous" and viral transformants, cancer cells), or in skin sections from aging subjects.

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Delta-Tocotrienol Causes Decrease of Melanin Content in Mouse Melanoma Cells

JOURNAL OF HEALTH SCIENCE ◽

10.1248/jhs.55.314 ◽

2009 ◽

Vol 55 (2) ◽

pp. 314-318 ◽

Cited By ~ 8

Author(s):

Akihiro Michihara ◽

Sachiyo Morita ◽

Yae Hirokawa ◽

Saya Ago ◽

Kenji Akasaki ◽

...

Keyword(s):

Melanoma Cells ◽

Melanin Content ◽

Mouse Melanoma

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Novel Chemically Modified Curcumin (CMC) Derivatives Inhibit Tyrosinase Activity and Melanin Synthesis in B16F10 Mouse Melanoma Cells

Biomolecules ◽

10.3390/biom11050674 ◽

2021 ◽

Vol 11 (5) ◽

pp. 674

Author(s):

Shilpi Goenka ◽

Francis Johnson ◽

Sanford R. Simon

Keyword(s):

Enzyme Activity ◽

Parent Compound ◽

Radical Scavenging ◽

Melanoma Cells ◽

Tyrosinase Activity ◽

Partial Recovery ◽

Tyrosinase Inhibitor ◽

Pyrocatechol Violet ◽

Mouse Melanoma ◽

Chemically Modified

Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation.

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Treatment with Uncaria tomentosa Promotes Apoptosis in B16-BL6 Mouse Melanoma Cells and Inhibits the Growth of B16-BL6 Tumours

Molecules ◽

10.3390/molecules26041066 ◽

2021 ◽

Vol 26 (4) ◽

pp. 1066

Author(s):

Ali Zari ◽

Hajer Alfarteesh ◽

Carly Buckner ◽

Robert Lafrenie

Keyword(s):

Tumour Size ◽

Melanoma Cells ◽

Tunel Staining ◽

Aqueous Extracts ◽

Ki 67 ◽

Uncaria Tomentosa ◽

Mouse Melanoma ◽

Anti Cancer

Uncaria tomentosa is a medicinal plant native to Peru that has been traditionally used in the treatment of various inflammatory disorders. In this study, the effectiveness of U. tomentosa as an anti-cancer agent was assessed using the growth and survival of B16-BL6 mouse melanoma cells. B16-BL6 cell cultures treated with both ethanol and phosphate-buffered saline (PBS) extracts of U. tomentosa displayed up to 80% lower levels of growth and increased apoptosis compared to vehicle controls. Treatment with ethanolic extracts of Uncaria tomentosa were much more effective than treatment with aqueous extracts. U. tomentosa was also shown to inhibit B16-BL6 cell growth in C57/bl mice in vivo. Mice injected with both the ethanolic and aqueous extracts of U. tomentosa showed a 59 ± 13% decrease in B16-BL6 tumour weight and a 40 ± 9% decrease in tumour size. Histochemical analysis of the B16-BL6 tumours showed a strong reduction in the Ki-67 cell proliferation marker in U. tomentosa-treated mice and a small, but insignificant increase in terminal transferase dUTP nick labelling (TUNEL) staining. Furthermore, U. tomentosa extracts reduced angiogenic markers and reduced the infiltration of T cells into the tumours. Collectively, the results in this study concluded that U. tomentosa has potent anti-cancer activity that significantly inhibited cancer cells in vitro and in vivo.

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