agar and Carcinoma--Squamous-Cell

We have shown earlier that overexpression of Calreticulin (CRT) contributed to a poor prognosis for patients with esophageal squamous cell carcinoma (ESCC). Here, we have shown an important role of CRT in tumorigenesis through enhancing cell motility and anoikis resistance. SiRNA-mediated knockdown of CRT caused impaired cell migration, invasion and resistance to anoikis. Notably, CRT downregulation decreased the expression of Cortactin (CTTN), which has been previously reported as a candidate oncogene associated with anoikis through the PI3K-Akt pathway. In addition, Akt phosphorylation was abolished after CRT downregulation and its activation can be refreshed by CRT upregulation, suggesting that CRT-enhanced cell resistance to anoikis through the CRT-CTTN-PI3K-Akt pathway. Moreover, the CTTN mRNA level was decreased in CRT-siRNA cells, coupled with the inactivation of STAT3. Expression of both CTTN and p-STAT3 was reduced in tumor cells following incubation with the JAK-specific inhibitor, AG490. Chromatin immunoprecipitation assay showed direct binding of p-STAT3 to the conservative STAT3-binding sequences in CTTN promoter. Furthermore, overexpression of CTTN in CRT-downregulated ESCC cells restored its motility and resistance to anoikis. This study not only reveals a role of CRT in motility promotion and anoikis resistance in ESCC cells, but also identifies CRT as an upstream regulator in the CRT-STAT3-CTTN-Akt pathway.

Topics: Agar; Animals; Anoikis; Calreticulin; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cortactin; Down-Regulation; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; STAT3 Transcription Factor; Transcription, Genetic

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin-protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G(1) progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-Ras(G12V) to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Topics: Agar; Animals; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; Epithelial Cells; Epithelium; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Microtubule-Associated Proteins; Mouth Neoplasms; Neoplasm Invasiveness; Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Rats; S-Phase Kinase-Associated Proteins; Transfection; Tumor Suppressor Proteins

The ultrastructure of cells from seven human lung cancers and from the colonies formed by these cells in soft agar was investigated. Tumor cells developed to display the morphofunctional potentials of the initial tumors. Cultured cells of squamous-cell carcinomas contained numerous tonofilaments, those of adenocarcinomas developed microvilli on their apical surfaces and intracellular lumens. On the other hand, cells of squamous-cell carcinomas showed features specific of adenoma epithelium, i.e. well developed microvilli and intracellular lumens. Besides, cells of adenocarcinoma often contained large quantities of tonofilaments considered to be characteristic of epidermoid epithelium. The results obtained suggest a possibility of metaplastic transformation of the lung epithelium.

Topics: Adenocarcinoma; Adenocarcinoma, Bronchiolo-Alveolar; Agar; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Culture Media; Humans; Lung Neoplasms; Methotrexate; Microscopy, Electron; Morphogenesis; Prospidium; Tumor Cells, Cultured; Vincristine

Clonogenic assays under either anchorage-dependent or -independent conditions are very useful for testing the sensitivity of tumor cells to cytotoxic drugs and radiation. These assays have not been widely used with squamous-cell carcinomas (SCC) because of poor tumor-cell viability and poor cloning efficiency, especially in semi-solid media. To find a clonogenic assay suitable for use with human squamous cancers we tested SCC lines, derived in our laboratory from patients with head and neck cancer, for the capacity to form colonies in soft agar and in 96-well plates. Of 13 UM-SCC lines tested for colony formation in agarose, only UM-SCC-11A was capable of growth in conventional semi-solid media. One other line, UM-SCC-14C, produced colonies in agarose only in the presence of epidermal growth factor. In contrast, all 17 of the SCC lines tested exhibited colony formation in adherent cell culture using limiting dilution in 96-well plates. The plating efficiencies of the SCC lines in the 96-well plate assay ranged from 0.02 to 0.52 colonies (wells)/cell whereas the PE values in soft agar were lower, ranging from 0.0055 to 0.0086 colonies/cell. The 96-well plate assay is not affected by cell migration, a problem encountered with some cell lines when clonogenic assays are performed in Petri dishes. UM-SCC-11A was tested for radiation sensitivity both in soft agar and in the 96-well plate assay. Comparable results were obtained. In summary, the majority of SCC cell lines did not form viable colonies in soft agar but the 96-well plate assay was applicable to a broad spectrum of anchorage-dependent human SCC cell lines and provides an efficient method for evaluating clonogenic cell survival.

Topics: Agar; Carcinoma, Squamous Cell; Cell Movement; Cell Survival; Colony-Forming Units Assay; Humans; Tumor Cells, Cultured; Tumor Stem Cell Assay

The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.

Topics: Agar; Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Peptides; Transforming Growth Factors; Tumor Cells, Cultured

Radiation survival curves have been generated for 3 human tumor cell lines as a means of comparing and evaluating the validity of human tumor soft-agar clonogenic assays. The assays investigated were the Hamburger-Salmon, Courtenay-Mills, Courtenay-Mills plus additions, soft agar (no additions), and soft agar plus additions. The additions were formulated to supplement the media used in soft agar assays of primary ovarian and cervical carcinoma specimens. Supplementing the media with additions led to a 2- to 3-fold increase in PE of CaSki cells but had no effect on the PEs of ME180 and OWI cells. Radiation survival curves were similar in all assays for CaSki and OWI but differed for ME180 cells. For ME180 cells, the Courtenay-Mills and soft agar assays plus additions produced the most radioresistant curves (Do = 2.2 Gy); the cells were more responsive when assayed by the Hamburger-Salmon method (Do = 1.5 Gy), and the soft agar and Courtenay-Mills assays gave the most radiosensitive curves (Do = 1.2 Gy). These results demonstrate that the PE of human tumor cell lines may be increased with no effect on radiation survival; radiation survival may be altered without changes in PE and neither may be altered by applying modifications and supplements to existing clonogenic assays.

Topics: Adenocarcinoma; Agar; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Colony-Forming Units Assay; Female; Humans; Neoplasms; Ovarian Neoplasms; Tumor Stem Cell Assay; Uterine Cervical Neoplasms

An in vitro human tumor cell assay was used in an attempt to culture head and neck tumors from patients with squamous cell carcinomas. Initially, specimens from nine head and neck tumors were disaggregated by mechanical methods and assayed in soft agar. Five of nine tumors grew in the soft-agar system yielding a cloning success rate of 56%. Plating of 5 X 10(5) cells resulted in 12 to 255 colonies per plate after 21 days in culture, with a cloning efficiency between 0.002% and 0.08%. Recently, the authors replaced the agar with an agarose culture matrix. Of 10 specimens with positive pathology, 9 have shown colony growth (greater than 20 cells). Cloning efficiency in agarose improved approximately 2-fold. Morphologic assessment of tumor colonies in culture showed the same characteristics as those of the original tumor. Overall success rate of growing head and neck tumors in agar and agarose has been 14 of 19 patients (74%). The development of a soft agarose assay for head and neck tumor cells should provide an in vitro technique for predicting in vivo response to anticancer drugs and other therapeutic modalities such as radiotherapy and hyperthermia.

Topics: Agar; Carcinoma, Squamous Cell; Cells, Cultured; Cytological Techniques; Head and Neck Neoplasms; Humans; Sepharose; Stem Cells

The factors controlling the growth of human tumor cells in soft agar are poorly understood. However, it has been demonstrated that serum provides factors which promote anchorage-independent growth. We tested 58 tumor specimens, which were obtained from patients with adenocarcinoma of the lung, colon, ovary or squamous cell carcinoma, for their ability to form colonies in soft agar in serum-free or serum-supplemented media. The cells were unable to replicate, and none of the hormones or growth factors tested: insulin (I), transferrin (T), selenium (S), estradiol (E), hydrocortisone (H) or epidermal growth factor (EGF) could substitute for serum. Examination of the serum dose-response curves indicated that growth factors reduced the serum concentrations needed to support anchorage-independent growth. The addition of the supplements and the lowering of serum concentrations increased cloning efficiencies (C.E.) in 38/51 trials, when cells were able to grow initially. The addition of ITS increased C.E. in 18/21 cases, HITES in 15/17 cases and EGF in 12/18 cases as compared to controls. ITS and HITES increased the number of colonies only when serum was the limiting factor. EGF, however, increased the number of colonies even when serum was not the limiting factor. The ability of the supplements to enhance growth could not be correlated to tumor type or initial cloning efficiencies. However, in only 1/25 cases were cells that were unable to form colonies under standard conditions induced to form colonies in the presence of the growth factors. Normal and tumor-derived human fibroblasts did not form colonies in soft agar in the presence of these growth factors. The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.

Topics: Adenocarcinoma; Agar; Blood; Carcinoma, Squamous Cell; Cell Division; Clone Cells; Colonic Neoplasms; Epidermal Growth Factor; Estradiol; Female; Humans; Hydrocortisone; Insulin; Lung Neoplasms; Ovarian Neoplasms; Selenium; Transferrin

Established cell lines from 8 human squamous cell carcinomas (SCC) together with normal human keratinocytes, have been investigated for their ability to grow in soft agar and as xenografts when injected as a single cell suspension into immunologically incompetent mice. One of 8 SCC lines formed colonies with efficiencies greater than 1% in soft agar, and only 2 formed progressively growing tumors when injected into animals. It is concluded that these 2 criteria are not reliable markers of malignant transformation in squamous epithelia unless cytological criteria are also applied.

Topics: Agar; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Immunocompetence; Mice; Mice, Nude; Neoplasm Transplantation; Skin

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

The growth characteristics of LT-2 cells, an epithelial squamous cell carcinoma, clearly separate the phenotypes of anchorage-independent growth in soft agar and tumor formation in vivo. LT-2 readily grows and forms tumor nodules in the nude mouse but does not proliferate anchorage independently in soft agar. This distinction is confirmed by the observation that cells explanted from nude mouse tumor nodules and cultured in vitro still do not clone in soft agar. The human origin of tumor nodule cells was confirmed by karyotyping. LT-2 cells have an aneuploid karyotype which has persisted for 4.5 years in culture with considerable variation in chromosomal number. Passage through the nude mouse did not select for any "tumor" clone since marked chromosomal variation was still noted by cells explanted from tumor nodules. Tumor cells formed a well-differentiated skin with keratin formation in the nude mouse despite wide karotypic variations of cells and years of in vitro culture. Strict monolayer growth was noted by LT-2 cells when grown in culture flasks and also by cells explanted from tumor nodules, indicating that monolayer growth and nude mouse tumorigenicity are also separate phenotypes.

Topics: Agar; Aneuploidy; Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cytological Techniques; Karyotyping; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Phenotype

Topics: Agar; Biopsy; Carcinoma, Squamous Cell; Cell Division; Cell Line; Clone Cells; Colonic Neoplasms; Head and Neck Neoplasms; Humans; Intestinal Neoplasms; Neuroblastoma; Rectal Neoplasms

The soft-agar stem-cell assay was applied to head and neck cancer. We have successfully grown 23 (64%) of 36 head and neck tumors from both primary lesions and metastases. More poorly differentiated tumors had positive cultures more frequently than well-differentiated tumors. The plating efficiency (colonies per cells in the inoculum) averaged 0.006% (range, 0.001% to 0.08%). The system allows testing and sensitivity of individual tumors to cancericidal drugs, and our initial trials using methotrexate, bleomycin sulfate, and cisplatin (cisplatinum) show a high degree of variability between individual tumors.

Topics: Agar; Antineoplastic Agents; Carcinoma, Squamous Cell; Cells, Cultured; Colony-Forming Units Assay; Drug Resistance; Head and Neck Neoplasms; Humans

Topics: Adenocarcinoma; Adult; Agar; Animals; Antineoplastic Agents; Anus Neoplasms; Breast Neoplasms; Carcinoma, Squamous Cell; DNA; Dogs; Humans; Leucine; Leukocytes; Liver; Mice; Middle Aged; Neoplasms; Neoplasms, Experimental; Parotid Neoplasms; Proteins; Radioisotopes; Rectal Neoplasms; RNA; Sulfhydryl Compounds; Thoracic Neoplasms; Thymidine; Uridine

Topics: Agar; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line; Culture Techniques; Diffusion; Humans; Indophenol; L Cells; Leukemia L1210; Methods; Neoplasms, Experimental