agar and Skin-Neoplasms

Paraffin embedding of small, thin tissue samples requires specific expertise for optimal orientation before tissue sectioning. This study evaluates the real-life utility of the agar pre-embedding technique for small skin biopsies with regards to lengthening of work times, problems in orientation (re-embedding) and ancillary techniques (immunohistochemistry and in situ hybridisation) between two high work flow pathology laboratories, one of which routinely uses the agar pre-embedding technique and one which does not. The mean time required for pre-embedding in agar was 30.4 s, but time for paraffin embedding for agar pre-embedded samples was shorter than the traditional method (177 vs 296 s; p<0.005). The number of skin samples requiring re-embedding was significantly higher with the traditional embedding method (p<0.005). No problems in immunoreactivity were observed in all 1900 reactions performed with 17 different antibodies. Fluorescence in situ hybridisation analysis was optimised with a prolonged protease K incubation time (21 vs 18 min).

Topics: Agar; Biomarkers, Tumor; Biopsy; High-Throughput Screening Assays; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Paraffin Embedding; Pathology, Clinical; Predictive Value of Tests; Reproducibility of Results; Skin; Skin Neoplasms; Time Factors; Workflow

We have previously reported that agaro-oligosaccharides (AGOs) suppressed the elevated levels of nitric oxide (NO), prostaglandin E₂(PGE₂), and pro-inflammatory cytokines in activated monocytes/macrophages, via heme oxygenase-1 induction. In this report, we initially demonstrated that AGOs intake inhibited NO production in activated peritoneal macrophages. Then, we tested for the ability of AGOs to prevent tumor promotion on the two-stage mouse skin carcinogenesis model. As a result, AGOs feeding led to delayed tumor appearance and decreased tumor number. It is known that PGE₂ is one of key players in carcinogenesis. Thus, we confirmed that PGE₂ production was suppressed by AGOs intake in TPA-induced ear edema model. We also demonstrated that cyclooxygenase-2 and microsomal PGE synthase-1, rate-limiting enzymes in PGE₂ production, were down-regulated by AGOs in human monocytes. Consequently, AGOs are expected to prevent tumor promotion by inhibiting PGE₂ elevation in chronic inflammation site.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Agar; Animals; Antineoplastic Agents; Carcinogens; Cyclooxygenase 2; Dinoprostone; Disease Models, Animal; Edema; Humans; Intramolecular Oxidoreductases; Leukocytes, Mononuclear; Macrophages, Peritoneal; Male; Mice; Mice, Inbred ICR; Nitric Oxide; Nitrites; Oligosaccharides; Prostaglandin-E Synthases; RNA, Messenger; Skin Neoplasms; Tetradecanoylphorbol Acetate

Utilizing gene microarray profiling of melanoma samples, we have recently identified a novel gene overexpressed in both thick primary and metastatic melanomas. This gene, progestagen-associated endometrial protein (PAEP), has never before been implicated in the oncogenic processes of melanoma, with its true function in oncogenesis and tumour progression relatively unknown. Overexpression of the PAEP gene in freshly procured thick primary and metastatic melanoma samples (58%) and daughter cell lines (77%) is confirmed by quantitative RT-PCR, immunohistochemistry, Western blotting and mass spectrometric analysis. We suggest that PAEP gene overexpression is involved with melanoma tumour progression as well as an aggressive phenotype. Transfection of melanoma cells with PAEP small interfering RNA (siRNA) reveals a significant decrease in soft agar colony formation and a marked inhibition of both cell migration and cell invasion. Furthermore, we establish stable melanoma transfectants via PAEP lentiviral small hairpin RNA (shRNA), examine their growth characteristics in a murine xenograft model and reveal that tumour growth is significantly inhibited in two separate melanoma cell lines. Our data strongly implicate the PAEP gene as a tumour growth promoter with oncogenic properties and a potential therapeutic target for patients with advanced melanoma.

Topics: Agar; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Glycodelin; Glycoproteins; Humans; Lentivirus; Melanoma; Mice; Neoplasm Invasiveness; Pregnancy Proteins; Reproducibility of Results; RNA, Small Interfering; Skin Neoplasms; Transcription, Genetic; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Losses of heterozygosity involving chromosomes 9 and 10 are frequent events in the development and progression of cutaneous malignant melanoma. To investigate whether specifically deleted chromosomal regions encode tumor suppressor genes (TSGs), we introduced normal chromosome 10 into the tumorigenic human metastatic melanoma cell line UACC-903 by microcell fusion. In addition, two chromosome 9 derivatives that were microdeleted in the region of the p16INK4A/p15INK4B locus were transferred to determine whether an additional melanoma TSG or TSGs reside on chromosome 9p, as indicated by previous melanoma allele loss studies. In comparison to parental cells, microcell hybrids generated with chromosomes 9 (microdeleted) and 10 displayed reduced anchorage-independent growth in soft agar and markedly reduced tumorigenicity in athymic (nu/nu) mice. These data define a TSG or TSGs that function independently of p15/p16 on chromosome 9 and provide evidence for a TSG (or TSGs) on chromosome 10 that may be important in melanoma development.

Topics: Agar; Animals; Carrier Proteins; Cell Cycle Proteins; Cell Division; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 10; Chromosomes, Human, Pair 9; Cloning, Molecular; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Female; Genes, p16; Genes, Tumor Suppressor; Humans; Melanoma; Mice; Mice, Nude; Neoplastic Stem Cells; Phenotype; Skin Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Proteins

In 23 patients, donor sites of split-thickness skin grafts were treated with Geliperm Hydrogel, a swellable polyacrylamide agar. Healing duration, toleration, exudation and pain were all noted during the daily change of dressing. In 22 of the 23 cases, good healing was obtained after an average of 12.3 days. We feel that Geliperm is excellently suitable for covering the donor sites of split-thickness skin grafts.

Topics: Acrylamides; Agar; Aged; Female; Humans; Male; Skin Neoplasms; Skin Transplantation; Surgical Flaps; Wound Healing

A single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) followed by twice-weekly 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment was given to the skin of female CD-1 mice, and soft agar colony formation of the epidermal cells was evaluated periodically during the treatment. From the second week, colony growth was observed and the number of colonies increased with time, reaching a significant level from the fourth week. Colony formation was observed earlier than papilloma development in the mouse skin. Soft agar colony-forming assay might be a useful tool to analyze the early events in carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Agar; Animals; Cocarcinogenesis; Colony-Forming Units Assay; Epidermis; Male; Mice; Papilloma; Skin Neoplasms; Tetradecanoylphorbol Acetate; Time Factors; Tumor Stem Cell Assay

Human tumors were cultured by the two-layer soft-agar technique and the time course of tumor colony development was evaluated during periods of up to 6 weeks in culture. All colony counting was performed with an automated tumor colony counter (Omnicon; Bausch and Lomb, Inc, Rochester, NY, USA). This instrument provided colony counts per culture plate in six size categories from greater than 60 microns diameter colonies to greater than 149 microns diameter colonies. Six to 24 culture plates were used for each "growth curve", generally 24. Control (non-drug-treated) cultures were obtained from 117 tumors, of which 25 also provided enough cells to allow evaluation of the time course of colony development after exposure to cytostatic agents. The development of colonies in non-drug-treated plates usually demonstrated a lag phase, a logarithmic growth phase to maximum colony development and a subsequent deterioration of colonies. In spite of clumps seeded into the agar, real colony growth could be recognized by frequent colony counting of culture dishes, although the temporal patterns of growth were sometimes different if pure single-cell suspensions were compared with suspensions containing clumps from the same tumor. Drug pre-incubation caused changes in the temporal pattern of colony growth as well as in the total number of colonies. Some cultures showed drug sensitivity when evaluated at certain time points while evaluation at later time points showed only borderline drug effect or none at all. The potential utility of tumor colony growth curves in the clinical applications of tumor colony cultures is discussed.

Topics: Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Small Cell; Colony-Forming Units Assay; Cystadenoma; Female; Humans; Male; Ovarian Neoplasms; Prostatic Neoplasms; Rats; Skin Neoplasms; Staining and Labeling; Time Factors; Tumor Stem Cell Assay

A previously unpublished reaction of precipitation in agarose between histone and non-histone proteins extracted in saline buffer from nuclei of human skin tumors, is reported. Two bands of precipitation similar to those in an immunodiffusion reaction between NHP and specific antisera, were observed. The reaction described is very similar to the affinodiffusion reaction of glycoproteins and lectins in agarose.

Topics: Agar; Animals; Carcinoma; Chromosomal Proteins, Non-Histone; Fibroma; Histones; Humans; Immunodiffusion; Melanoma; Rats; Rats, Inbred Strains; Skin Neoplasms

Colony growth in soft agar of human melanoma cells from biopsy material, cell lines and xenografts was evaluated. Colony forming potential is constantly very low in all studied types of tumor tissue, however slight increase in clonogenic potential was seen during subsequent xenograft passages, but within the range of less than 1%. Results of presented studies suggest that the number of colonies does necessarily express the degree of colony forming potential and the culture preparation and conditions as well. It appears that there is no reliable correlation between the number of colonies in soft agar and the clinical course of melanoma patients under study.

Topics: Agar; Animals; Biopsy; Cells, Cultured; Humans; In Vitro Techniques; Melanoma; Mice; Skin Neoplasms; Transplantation, Heterologous; Tumor Stem Cell Assay

An in vitro soft agar technique was used to culture human malignant melanoma cells from 61 solid tumors, 17 lymph nodes, 11 effusions, and four bone marrow specimens from 93 patients with malignant melanoma. Colonies grew in soft agar from 64 (69%) of the 93 specimens. Fifty-five percent of the specimens cultured formed greater than or equal to 30 colonies per 500,000 nucleated cells plated. Light microscopy, electron microscopy, tumor marker, and athymic nude mouse studies provided evidence the colonies were composed of malignant melanoma cells. Drug sensitivity studies utilizing the cloning technique showed similarities between in vitro results and the general clinical experience noted with the same drugs. The human tumor cloning system represents a new model for future basic biology and clinical studies of human malignant melanoma.

Topics: Agar; Animals; Antineoplastic Agents; Cell Count; Cell Survival; Clone Cells; Cytological Techniques; Drug Evaluation, Preclinical; Humans; Melanoma; Mice; Mice, Nude; Skin Neoplasms; Time Factors

Topics: Agar; Bone Marrow; Carcinoma, Squamous Cell; Clone Cells; Cytoplasm; Desmosomes; Head and Neck Neoplasms; Humans; Melanoma; Microscopy, Electron; Neuroblastoma; Skin Neoplasms

An in vivo-in vitro implantation model has been used to investigate further the early stages of chemically induced s.c. neoplasia in the mouse. Cell cultures of implant-site tissues from control and 3,4-benzpyrene (BP)-treated animals were found to mirror the in vivo tissue reactions occurring at the time of explantation (Westwood et al., 1979). Cells were classified into 6 different types. The most abundant cell type in later control cultures was of a typical fibroblast morphology. However, a suppression of growth of fibroblast-like cells occurred when BP-treated tissues were explanted, and a selection of growth in favour of the large polygonal Type 5 cells was observed. When grown from BP-treated tissues Type 5 cells were found to be capable of growth in a semi-solid agar medium. Quantitative studies showed that cells capable of growth in agar reached a peak about 4 weeks after implantation, followed by a decline in numbers until the formation of tumours. This observation may result from the parameters regulating the development of chemically induced neoplasia in the subcutis.

Topics: Agar; Animals; Basophils; Benzopyrenes; Cells, Cultured; Female; Fibroblasts; Mice; Neoplasms, Experimental; Skin Neoplasms; Time Factors

Topics: Agar; Animals; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transplantation, Isogeneic

A cell line, derived from a spontaneous equine connective tissue tumor (equine sarcoid), has been established. The morphological and growth characteristics indicative of malignant transformation of the cells include a disoriented, rapid growth and loss of contact inhibition. Further evidence of transformation is the agglutination of these cells by concanavalin A and their ability to divide in semisolid media.

Topics: Agar; Agglutination Tests; Animals; Cell Line; Cell Transformation, Neoplastic; Concanavalin A; Culture Media; Culture Techniques; Horses; Male; Skin Neoplasms; Trypsin

Topics: Adolescent; Adult; Agar; Aged; Catechol Oxidase; Child; Child, Preschool; Dihydroxyphenylalanine; Electrophoresis; Female; Gels; Humans; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Male; Melanoma; Middle Aged; Nevus, Pigmented; Skin; Skin Neoplasms; Tissue Extracts; Tyrosine

Topics: Agar; Clinical Laboratory Techniques; Collagen Diseases; Dermatology; Humans; Laboratories; Skin Neoplasms