Carcinoma Cells In Mice Research Articles

Effect of cidofovir on the tumor growth of SiHa (human cervical carcinoma) cells in nude mice

Effect of cidofovir on the tumor growth of SiHa (human cervical carcinoma) cells in nude mice

Impact of the cyclin-dependent kinase inhibitor p27Kip1 on resistance of tumor cells to anticancer agents.

A low proliferating fraction in solid tumors limits the effectiveness of cell cycle-dependent chemotherapeutic agents. To understand the molecular basis of such "kinetic" resistance we cultured tumor cells as multicellular spheroids and examined levels of p27Kip1, a cyclin-dependent kinase inhibitor known to be upregulated by intercellular contact in normal cells. When transferred from monolayer to three-dimensional culture, a consistent upregulation (up to 15-fold) of p27 protein was observed in a panel of mouse and human carcinoma cell lines. Antisense-oligonucleotide-mediated downregulation of p27 in EMT-6 mammary tumor cell spheroids reduced intercellular adhesion, increased cell proliferation, sensitized tumor cells to 4-hydroperoxycyclophosphamide, and restored drug- or radiation-induced cell-cycle perturbations repressed in spheroid culture. Our results implicate p27 as a regulator of drug resistance in solid tumors and suggest that tumor-targeted p27 antagonists may be useful chemosensitizers in conjunction with conventional anticancer therapy.

Interactions between CD44 and hyaluronic acid: their role in tumor growth and metastasis.

Adhesion molecules enable lymphocytes to interact with antigen-presenting cells or target cells more effectively (Springer 1990; Butcher 1986; Stoolman 1989). Adhesion molecules also allow lymphocytes or monocytes to interact with endothelial cells or high endothelial venules (HEV). Adhesion molecules enable lymphocytes or monocytes to recirculate and home to specific anatomical sites during inflammation. Tumor metastasis is a complex phenomenon involving a sequence of events that remain poorly understood (Fidler 1978; Nicolson 1988; Kahn 1992; Stetler-Stevenson et al. 1993; Turley 1984). Tumor metastatic cascade involves tumor cell and host cell interactions and may also involve interactions among tumor cells. Tumor cells developed in different microenvironments may utilize different mechanisms for invasion and metastasis. This interpretation was supported by results obtained from experiments using orthoptopic implantation of human carcinoma cells in nude mice (Fidler and Radinsky 1990; Fidler et al. 1990). Human colon carcinoma cell lines do not metastasize unless they are injected into the cecum or spleen of nude mice (Fidler 1991). Subcutaneous injection of the same tumor cell lines results in a significantly lower frequency of metastasis formation. These observations have also been observed with human breast, stomach, pancreas, and prostate tumor cell lines (Fidler and Radinsky 1990; Fidler 1990, 1991). In general, orthotopic transplants of human tumors into nude mice favors recapitulation of the metastatic patterns seen in patients with the same tumor.

Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes.

Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.

Apoptosis of stomach carcinoma cells induced by a human monoclonal antibody

Although stomach carcinoma is estimated to be one of the most frequent cancers worldwide, still little is known about the immunity of patients with stomach cancer. Humoral tumor immunity has been studied by isolating B cells of patients with cancer to characterize the activity of such antibodies on tumor cells. Apoptosis, programmed cell death, explains the suicide of cells by fragmentation of DNA, cell shrinkage and dilation of endoplasmatic reticulum, followed by cell fragmentation and formation of membrane vesicles called apoptotic bodies. Apoptosis serves to remove unwanted, damaged, or dangerous, e.g., precancerous cells. The human monoclonal antibody SC-1 was isolated from a patient with a signet ring cell carcinoma of the stomach by fusion of spleen lymphocytes to the heteromyeloma SPM4-0. The antibody was tested for growth-inhibiting effects in vitro in soft agar assays, in 3-H thymidine uptake experiments, and in a mitochondrial enzymatic activity assay. In vivo intraperitoneal tumor growth was investigated in nu-nu mice. The immunoglobulin M (lambda) antibody identifies a molecule with a molecular weight of approximately 49 kilodaltons in stomach carcinoma cells. No reactivity was observed with carcinomas of other origins, melanomas, lymphomas, or normal tissue. When tested in vitro, the antibody inhibited tumor cell growth in cell culture and soft agar. In vivo growth of stomach carcinoma cells in nu-nu mice was reduced when the antibody was injected after the tumor cells. Ultrastructural and functional studies revealed that the SC-1 antibody induced apoptosis of tumor cells. The human monoclonal antibody SC-1 described here inhibited growth of stomach carcinoma cells in vitro and in vivo by inducing apoptosis, and may, therefore, be valuable for treating patients with stomach carcinoma.

Spontaneous metastasis of PC-3 cells in athymic mice after implantation in orthotopic or ectopic microenvironments.

The ability of subcutaneous, prostatic, and nonprostatic intraabdominal organ microenvironments to influence local tumor growth and metastasis of PC-3 human prostate carcinoma cells in athymic mice was determined. Tumorigenesis and metastasis of PC-3 were evaluated 60 days after subcutaneous and intraprostatic (orthotopic) implantation of 5 x 10(5) PC-3 cells in 6-week-old, male athymic mice. Intraprostatic implantation of PC-3 cells resulted in paraaortic lymph node metastases in 10 of 10 (100%) mice with prostatic tumors, whereas metastases were present in only 2 of 9 (22%) mice after subcutaneous implantation. Next, we determined whether the urinary bladder (nonprostatic, urogenital microenvironment) or stomach (nonurogenital, intraabdominal microenvironment) would facilitate the metastasis of PC-3 cells in athymic mice. Tumorigenesis and metastasis were 100% after subserosal implantation of PC-3 cells within the wall of the urinary bladder (n = 6 mice). Subserosal implantation of PC-3 cells into the stomach wall (n = 7 mice) also resulted in tumor formation and metastasis to regional lymph nodes in 100% of mice. In all experiments, regional lymph nodes were the most frequent site of metastasis, regardless of implantation site. We conclude that tumor microenvironment factors responsible for the metastasis of PC-3 cells in athymic mice may not be organ-specific, since nonprostatic visceral microenvironments are sufficient for predictable metastasis. Use of these models may further our understanding of how tumor microenvironment modulates expression of the metastatic phenotype by human prostate carcinoma cells.

Mode of action of photodynamic therapy with sulfonated aluminum phthalocyanine in induced squamous cell carcinomas in animal models.

In order to investigate the mechanism of action of photodynamic therapy (PDT) with sulfonated aluminum phthalocyanine (AlSPc) in squamous cell carcinoma, animal experiments were performed in induced carcinomas of the mucosa of the hamster's cheek pouch and skin of the laboratory mouse. Histological examinations revealed signs of massive interstitial bleeding, indicating a vascular response to PDT with AlSPc. It was also possible to induce similar change adjacent to newly formed vessels at the margin of an inflammatory reaction in the cheek pouch of five hamsters in the absence of tumor cells. Implanted human squamous cell carcinoma cells in athymic nude mice showed that carcinoma cells removed immediately following PDT remained viable, while tumors left in situ became necrotic. These results suggest that the primary effect of PDT with AlSPc in vivo is not the malignant cell itself, but the vascular stroma of the tumor or in the immediate vicinity of the latter.

DLC1 suppresses distant dissemination of human hepatocellular carcinoma cells in nude mice through reduction of RhoA GTPase activity, actin cytoskeletal disruption and down-regulation of genes involved in metastasis

The process of cell dissemination from the primary tumors to distant sites is the most harmful event during cancer progression, and the leading cause of cancer death. We have previously demonstrated that restoration of DLC1 tumor suppressor gene expression in the DLC1-negative Focus and 7703K human hepatocellular carcinoma (HCC) cell lines induced caspase-3 mediated apoptosis, reduced cell growth in vitro and tumorigenicity in vivo and diminished the ability to migrate through Matrigel, a property suggestive of metastatic potential in vivo. We now show that subcutaneous tumors developing after inoculation of Focus and 7703K cells into nude mice disseminate cells to liver and lung, and this process is markedly suppressed by restoration of DLC1 expression. Inhibition of tumor cell dissemination was associated with lower levels of RhoA activity, an increase in rounded cells and a reduction in actin stress fibers and focal adhesion molecules that are of critical importance in cancer cell invasion and metastasis. In addition, DLC1 down-regulated the expression of osteopontin and matrix metalloproteinase-9, which are highly up-regulated in most primary HCC with associated metastases. These observations implicate the DLC1 gene in suppression of HCC cell dissemination and identify novel cellular and genetic alterations that contribute to prevention of metastasis, a life-threatening event in cancer progression.

Endothelial cells enhance spontaneous metastasis of human lung carcinoma cells in athymic mice : Varki NM, Viswanathan B, Vu T. Department of Medicine, Cancer Biology Program of the University of California, San Diego, La Jolla, CA 92093. Cancer Lett 1990;51:251–257

Endothelial cells enhance spontaneous metastasis of human lung carcinoma cells in athymic mice : Varki NM, Viswanathan B, Vu T. Department of Medicine, Cancer Biology Program of the University of California, San Diego, La Jolla, CA 92093. Cancer Lett 1990;51:251–257

Quantitative microscopy of mouse colon 26 cells growing in different metastatic sites

Quantitative microdensitometry and computerised interactive image analysis were used to compare the expression of endogenous lectins by cells of mouse colon 26 carcinomas, growing either as primary tumours or metastases, in five different anatomic sites (caecum, liver, lung, spleen, s.c.). Endogenous lectins were visualised in tissue sections using the ABC peroxidase technique with a panel of 17 biotinylated neoglycoproteins representing a variety of carbohydrates found in glycoproteins, glycolipids and proteoglycans. Clear-cut site-associated differences in endogenous lectin expression were detected in cancer cells growing in all five sites. The patterns of these changes were complex and shifts in expression of different lectins were independently variable in both direction and amount. In addition to site-associated variations, differences in lectin expression were also detected in the liver and lungs, between cells in spontaneous metastases and cells in colonies generated by direct injection of cancer cells into the bloodstream. The results demonstrate quantitative, as distinct from qualitative, differences developing in cancer cell populations after delivery of cells to different target organs. The differences between liver and lung metastases are in accord with analogous site-associated differences in metastatic patterns produced by colon carcinoma cells in mice and in humans.

Influence of Organ Environment on Extracellular Matrix Degradative Activity and Metastasis of Human Colon Carcinoma Cells

Orthotopic implantation of human colon carcinoma cells is useful for studying the behavior of metastatic subpopulations. We observed that the parental line and variants of human colon carcinoma KM12 cells were all tumorigenic following implantation into the subcutis or cecal wall of BALB/c nude mice. Their ability to metastasize to distant organ sites varied, however, with the site of growth. Subcutaneous (SC) tumors did not produce visceral metastases, whereas cecal tumors metastasized to the regional mesenteric lymph nodes and to the liver. To examine the influence of organ environment on the extracellular matrix-degrading activity of the tumors, we inoculated human colon carcinoma cells into the subcutis or cecal wall and after 7 weeks isolated and cultured the tumors in serum-free medium. The conditioned media of SC tumors contained very low levels of type IV collagenase (gelatinase) and heparanase (heparan sulfate-specific endo-beta-D-glucuronidase), whereas the media of the cecal wall tumors contained high levels of both. Zymograms of the media revealed that the intracecal human colon carcinomas secreted more than three times the amount of latent and active forms of 92-kd type IV collagenase than did the SC tumors. Moreover, only the conditioned media of intracecal tumors contained latent and active forms of 64-kd type IV collagenase. Histochemical analysis using rabbit antiserum raised against the synthetic peptides of 72-kd procollagenase type IV showed type IV collagenase in the intracecal tumors; human colon carcinoma growing SC, however, were not stained significantly. These results suggest that factors in the organ environment may affect production and secretion of tumor extracellular matrix-degrading enzymes, and these factors may modify the metastatic behavior of human colon carcinoma cells in nude mice.

Endothelial cells enhance spontaneous metastasis of human lung carcinoma cells in athymic mice

The process of adhesion to endothelial cells is an important step in the progression to metastatic disease. The use of human neoplastic cell lines (now increasingly used in the study of the mechanisms of metastasis) in studying interactions with normal endothelial cells is therefore pertinent. In this report, the enhanced ability of a metastatic variant, MV522, of a human lung carcinoma cell line to adhere to endothelial cell monolayers is demonstrated. The ability to spontaneously metastasize from subcutaneous sites in athymic mice, of in vitro selected endothelial adhesive subpopulations of MV522 is also shown.

The α6β1 (VLA-6) and α6β4 protein complexes: tissue distribution and biochemical properties

A member of the integrin family, the alpha 6 beta 4 complex was previously identified on human and mouse carcinoma cell lines by using a rat monoclonal antibody to alpha 6. Here we describe two monoclonal antibodies that recognize epitopes on the beta 4 subunit of the human and mouse alpha 6 beta 4 complexes. The monoclonal antibodies against beta 4 were able to preclear alpha 6 beta 4, but not alpha 6 beta 1 from cell line extracts. A substantial fraction of the total beta 4 subunits present on the cell surface was not associated with alpha 6, as it could not be removed by anti-alpha 6 antibodies, but remained precipitable with anti-beta 4 antibodies. There was no evidence for novel alpha subunits associated with beta 4. The alpha 6 subunit consists of disulfide-linked heavy and light chains. The variability in size of these two chains from different cell types is largely due to differences in modifications of N-linked glycans. Additional heterogeneity may be caused by differential proteolytic cleavage of the alpha 6 precursor. Immunoperoxidase staining of tissue sections of neonatal and adult mice revealed that beta 4 expression is limited to epithelial tissues and peripheral nerves. The alpha 6 subunit has a wider distribution that includes all tissues and cells stained by antibodies against beta 4. Cells and tissue that are positive for alpha 6, but negative for beta 4, may express the alpha 6 beta 1 complex.

Melanin photosensitizes ultraviolet light (uvc) dna damage in pigmented cells

Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals.

Efficacy of antibodies to epidermal growth factor receptor against KB carcinoma in vitro and in nude mice.

Iodine-125-labeled monoclonal antibody 108.4 (108.4 mAb), raised against the extracellular domain of the epidermal growth factor (EGF) receptor, was shown to visualize sc xenografts of human oral epidermoid carcinoma (KB) cells in nude mice. In vitro, although EGF caused an increase in the number of KB cell colonies (150% at a concentration of 160 mM), the anti-EGF receptor antibodies reduced clone formation. At a concentration at which EGF caused a 50% increase in colony number, the addition of a 100-fold molar excess of 108.4 mAb resulted in a decrease in the number of cell colonies to 20% of the original value. Therefore, the effect of antibody on the KB tumor was studied in vivo in three different modes of tumor transplantation. Antitumor activity was demonstrated first by retardation (versus controls) of the growth of tumor cells as sc xenografts (P greater than .017), then by prolongation of the life span of animals with the ip form of the tumor (P less than .001), and finally on an experimental lung metastasis by a reduction in the number and size of tumors (P less than .05). When the anti-EGF receptor antibodies were added together with cisplatin, the antitumor effect was greatly enhanced, suggesting that the toxic activity of these agents is synergistic (P less than .007). The antitumor effect persisted when animals were treated with the F(ab)'2 fragment of the antibody, although it was less efficient. The Fab fragment of the antibody, whose ability to bind to the cell-associated receptor was completely conserved, did not affect the growth of the tumor. The activity manifested by the F(ab)'2 fragment of the anti-EGF receptor antibodies suggested that the antitumor effect was not due to immune mechanisms requiring the Fc portion of the antibody.

Invasion and metastasis of SY86B human gastric carcinoma cells in nude mice.

A moderately differentiated tubular adenocarcinoma of human stomach, named SY86B, was successfully transplanted subcutaneously to nude mice of different genetic backgrounds (BALB/CA/PBI‐nu, C57BL/6J.615/PBI‐nu and ICR‐BALB/CA/PBI‐nu). The tumor has been passaged for 13 generations and the transplantability was 100%. The SY86B cells retained the capacity of invasive and metastatic growth in the nude mice and showed a high rate of metastasis to the regional lymph nodes and to such distant organs as the lungs, liver and pancreas. The overall rate of metastasis was 77.7%. The species of the nude mice, their age and sex apparently did not significantly affect the occurrence of metastasis. Tumor‐bearing time and the aggressive character of the tumor cells themselves appeared important for the genesis of metastasis. This experimental model can provide a new approach to basic and clinical studies of cancer metastasis.

Testosterone-induced responsiveness to androgen in Shionogi mouse carcinoma cells

Primary cell cultures from an androgen-dependent mouse mammary carcinoma, the Shionogi-SC 115 tumor, were cultured in the presence or absence of testosterone (50 nM). Characteristic changes in cellular morphology and cell growth were observed according to the presence or absence of the androgen. In testosterone-containing medium, cells formed individual clones, piling up one over another and showed no contact inhibition, whereas in the absence of the androgen, cells had a flattened morphology, they grew in a monolayer and cell multiplication was reduced. The testosterone-dependent changes were observed in culture as long as cells were maintained in androgen-containing medium. Only a few (3–5) days of culture in the absence of testosterone rendered cells irreversibly unresponsive to the androgen, and they could no longer produce tumours after inoculation in the host animal. Cellular proteins were analysed after culture in the presence or absence of testosterone. After [ 35S]methionine labelling of cells and SDS-PAGE of the cytosol, several proteins were specifically synthesized in the presence of testosterone, predominantly a 45 kD protein, which was not seen in the absence of the androgen. Conversely, a protein of 35 kD present in absence of the hormone disappeared in the presence of testosterone. The anti-androgen cyproterone acetate inhibited the characteristic cellular morphology, cell proliferation and protein synthesis observed in the presence of the androgen. The antiprogestin and anti-glucocorticosteroid RU 486 also showed limited anti-androgen activity. The concentration of specific androgen receptor-binding sites did not change significantly after 3 months of culture with or without testosterone, i.e., in responsive and unresponsive cells.

Development of androgen resistance in mouse mammary tumor cells can be prevented by the antiandrogen flutamide.

As has been clearly demonstrated in prostate and breast cancer, progression to hormone insensitivity is a major problem responsible for the usually partial and short-lived response to antihormonal therapy. Preincubation of androgen-sensitive Shionogi mouse carcinoma cells for 15 days in the absence of androgens causes the development of complete resistance of cell growth to androgens. Of potentially important therapeutic significance is the finding that androgen sensitivity can be maintained not only by the androgen dihydrotestosterone (DHT) but also by incubation with the pure antiandrogen Flutamide-OH in the absence of androgens. Since androgen resistance is one of the main problems facing the treatment of prostate cancer, the possibility of avoiding or at least delaying the development of androgen resistance with a pure antiandrogen could well provide the basis for improving the success of therapy for this disease.

Enhanced spontaneous metastasis of mouse carcinoma cells transfected with an activated c-Ha-ras-1 gene.

To investigate whether the presence of an activated ras oncogene influences the ability of tumour cells to metastasize, the c-Ha-ras-1 oncogene cloned from EJ/T24 cells was introduced into MT1 Cl.5/7 mouse mammary carcinoma cells. Since the MT1 Cl.5/7 cells are already tumorigenic but have a low metastatic capacity, this experimental design allows a distinction to be made between the effects of the ras gene on metastasis and tumorigenicity. MT1 Cl.5/7 containing the EJ c-Ha-ras-1 metastasized more readily and to more tissue sites than control cells (2.8 sites/mouse vs 0.9 sites/mouse). The metastases expressed the EJ c-Ha-ras-1 p21 ras proteins; however, one metastasis was discovered that had lost the expression of the c-Ha-ras-1 gene. When these cells were re-tested for metastasis, the rate of metastasis was indistinguishable from that of controls. This observation, coupled with a demonstration that lung colonization potential following intravenous inoculation is unaffected by the presence of the activated ras gene, argues that the effect of mutant ras genes is exerted on the ability of cells to escape from the primary tumour, rather than on a survival in the circulatory systems and ability to seed a second site.

Site-dependence of invasiveness of ECA109 human oesophageal carcinoma cells in nude mice.

ECA109 human oesophageal carcinoma cells were injected either subcutaneously or intraperitoneally into BALB/CATc 1-nu/nu mice. After 23 weeks tumours were examined histologically and by scanning electron microscopy. Subcutaneous ECA109 tumours were well-delineated without signs of invasion. By contrast, intra-abdominal tumours invaded into the abdominal wall and abdominal organs. This result provides us with another example of site-dependence of invasion in vivo.