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Biomedical subjects

A F Chambers

Publications and source records attributed to A F Chambers.

At least 73 records · Page 4Linked to original sources
PubMed1996

Osteopontin expression in lung cancer.

Osteopontin (OPN), an integrin-binding, transformation-associated protein, is secreted by tumor cell lines in culture and is associated with increased malignancy in some experimental tumor systems. Little is known, however, about the significance of OPN expression in human cancers. The aims of this study were to determine if OPN was expressed in a series of surgically resected lung cancers, and if there was a relationship between OPN expression and clinico-pathologic findings or outcome. Twenty-five patients who underwent curative pulmonary resection were studied prospectively. RNA was extracted from primary tumor and distant normal lung tissue for each patient. OPN RNA levels were evaluated by northern blotting. Immunohistochemistry on paraffin-embedded tissue, using an anti-OPN monoclonal antibody, was performed to assess tissue distribution of OPN protein. OPN RNA and protein were over-expressed in the majority of tumors, relative to paired normal tissue. There was variation in the cells of the tumor that were OPN-immunopositive. In some cases OPN was present in tumor cells, while in the majority of cases OPN was detected primarily in tumor-infiltrating macrophages and necrotic areas. Over-expression of OPN RNA or protein generally was not related to clinico-pathological findings. However, there was a statistically significant association between OPN-immunopositivity in the tumor and patient survival. These findings suggest that OPN levels in lung tumors have the potential to provide clinically important predictive information on patient outcome, and that OPN may play a role in the biology of lung cancer.

Adult↗
PubMed1996

The predictive power of semiquantitative immunohistochemical assessment of p53 and c-erb B-2 in lymph node-negative breast cancer.

A series of 107 lymph node-negative (LNN) breast cancers was stained immunohistochemically with a combination of p53 and c-erb B-2. The immunohistochemical results were semiquantitated using a previously described system by Allred et al. p53 immunopositive cases were further screened for DNA mutations by the polymerase chain reaction-single-strand conformation polymorphism method (PCR-SSCP). Three representative cases showing mobility shifts were directly sequenced. One hundred of 103 invasive carcinomas were of no special type (infiltrating ductal carcinomas not otherwise specified). The three special type carcinomas included a tubular carcinoma, a classic infiltrating lobular carcinoma, and a mucinous carcinoma. Twenty-six patients (25.2%) had grade I carcinomas, and 77 patients (75%) had grade 2 or 3 carcinomas. There were four cases composed predominantly of ductal carcinoma in situ (DCIS) with foci of microinvasion. Twenty-seven of 107 patients (25%) died of disease. All those who died had grade 2 or 3 tumors. Univariate analysis showed that p53 and c-erb B-2 positivity (score > 6) were associated with a decreased overall survival (OS) (P = .0012 and P = .010, respectively), and a decreased disease-free survival (DFS) (P = .0009 and P = .027, respectively). The multivariate model selected these two variables as the best predictors of both OS and DFS (all P = or < .01). These results suggest that semiquantitative immunohistochemical analysis of p53 and c-erb B-2 provides prognostic information in LNN disease.

Adult↗
PubMed1996

Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices.

Two citrus flavonoids, hesperetin and naringenin, found in oranges and grapefruit, respectively, and four noncitrus flavonoids, baicalein, galangin, genistein, and quercetin, were tested singly and in one-to-one combinations for their effects on proliferation and growth of a human breast carcinoma cell line, MDA-MB-435. The concentration at which cell proliferation was inhibited by 50% (IC50), based on incorporation of [3H]thymidine, varied from 5.9 to 140 micrograms/ml for the single flavonoids, with the most potent being baicalein. IC50 values for the one-to-one combinations ranged from 4.7 micrograms/ml (quercetin + hesperetin, quercetin + naringenin) to 22.5 micrograms/ml (naringenin + hesperetin). All the flavonoids showed low cytotoxicity (> 500 micrograms/ml for 50% cell death). Naringenin is present in grapefruit mainly as its glycosylated form, naringin. These compounds, as well as grapefruit and orange juice concentrates, were tested for their ability to inhibit development of mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats. Two experiments were conducted in which groups of 21 rats were fed a semipurified diet containing 5% corn oil and were given a 5-mg dose of DMBA intragastrically at approximately 50 days of age while in diestrus. One week later, individual groups were given double-strength grapefruit juice or orange juice or fed naringin or naringenin at levels comparable to that provided by the grapefruit juice; in the second experiment, the rats were fed a semipurified diet containing 20% corn oil at that time. As expected, rats fed the high-fat diet developed more tumors than rats fed the low-fat diet, but in both experiments tumor development was delayed in the groups given orange juice or fed the naringin-supplemented diet compared with the other three groups. Although tumor incidence and tumor burden (grams of tumor/rat) were somewhat variable in the different groups, rats given orange juice had a smaller tumor burden than controls, although they grew better than any of the other groups. These experiments provide evidence of anticancer properties of orange juice and indicate that citrus flavonoids are effective inhibitors of human breast cancer cell proliferation in vitro, especially when paired with quercetin, which is widely distributed in other foods.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Tumor angiogenesis predicts recurrence in invasive colorectal cancer when controlled for Dukes staging.

Tumor angiogenesis is associated with metastasis in several types of solid tumors, including melanoma, breast, prostate, lung, bladder, and oral-cavity tumors. The purpose of this study was to determine whether tumor angiogenesis could predict recurrence following curative surgery for colorectal cancer. Thirty-five patients were studied, including 13 patients with recurrent tumor and 22 without. Representative formalin-fixed, paraffin-embedded sections of invasive colorectal cancers from these patients were sectioned. The endothelial cells of microvessels within the tumors were highlighted by immunohistochemical staining for CD31. The most active areas were identified and the microvessels counted in a x 400 field (0.152 mm2) by two observers in a blinded fashion. Tumor microvessel count (p = 0.0062). Dukes' staging (p = 0.0004), vascular invasion (p = 0.0280), and tumor grade (p = 0.0559) were all significantly associated with tumor recurrence. Tumor microvessel counts > or = 65 per x 400 field were associated with tumor recurrence (p = 0.0035, relative risk [RR] = 11.3). Controlling for Dukes' stage, a multivariate logistic regression model revealed that a tumor microvessel count > or = 65 is an important predictor of tumor recurrence (p = 0.0783, RR = 6.0). A backwards elimination proportional hazards model revealed that a microvessel count > or = 65 shows a trend toward independent prediction of time to tumor recurrence (p = 0.1203, RR = 2.967) when controlled for Dukes' staging (p = 0.0029, RR = 9.089). Despite the small number of patients studied, these results suggest that the number of microvessels in sections of invasive colorectal adenocarcinoma immunohistochemically stained with CD31 may be an important independent predictor of tumor recurrence and time to recurrence.

Adenocarcinoma↗
PubMed1995

Alternative splicing of PSP94 (prostatic secretory protein of 94 amino acids) mRNA in prostate tissue.

While performing reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total mRNA from prostate cancer specimens, two forms of PSP94 cDNA were detected. RT-PCR products were analysed by Southern blotting and probing with exon-specific oligonucleotides. In the short form of PSP94 mRNA, designated as PSP57, exon III was found to be deleted. The two mRNA forms were confirmed by cloning and sequencing of the RT-PCR products and were found to result from alternative splicing. The alternatively spliced form, PSP57, was characterized by sequence analysis. PSP94 and PSP57 possess identical exons I and II, including identical secretion signal peptide and the 5' untranslated sequences. PSP57 has a frame-shifted exon IV and encodes a putative 57 amino acid protein with a novel, highly basic C-terminus of 41 amino acids. PSP57 mRNA was detected in other urogenital tissues (kidney, bladder) and in most tumor cell lines tested, but was not detectable in other tissues such as breast and lung. In prostate tumor cell lines, PSP57 mRNA was aberrantly spliced and localized in the nuclear fraction of the cell. Our results suggest the possible existence of a novel PSP protein that originates from alternative splicing of PSP94 mRNA in urogenital tissues.

Aged↗
PubMed1995

Fate of melanoma cells entering the microcirculation: over 80% survive and extravasate.

Metastasis is an inefficient process; only a few cancer cells are able to form tumors after being released into the circulation. We studied the fate of cancer cells after injection into the circulation, quantifying their survival and ability to extravasate by 1 day later. B16F10 cells, parental or transfectants overexpressing tissue inhibitor of metalloproteinases 1, were injected i.v. into chorioallantoic membrane of chick embryos and analyzed by intravital videomicroscopy. Cell survival was quantified in two ways: (a) 15-microns microspheres were injected with cancer cells, and proportions of viable cells to microspheres were compared before and after injection; and (b) individual cancer cells were monitored continuously for 0.5-8-h intervals covering the first 24 h. Both methods showed virtually no destruction of cells. Greater than 80% of injected cells survived and extravasated by 24 h, indicating that growth after extravasation is a key stage of metastatic control.

Allantois↗
PubMed1995

Regulation and function of osteopontin in ras-transformed cells.

Transformation of nontumorigenic NIH 3T3 fibroblasts with an activated ras oncogene produces malignant cells that can metastasize. This induction of malignant behavior is due to changes in gene expression induced by Ras-mediated signal transduction. Osteopontin expression is induced in response to Ras, due to increased osteopontin transcription mediated by Ras-responsive regions in the osteopontin gene promoter. The increased expression of osteopontin contributes functionally to the malignant ability of the cells. Ras-transformed cells that express antisense osteopontin RNA show markedly reduced ability to form tumors and to metastasize in experimental animals. Increased osteopontin expression in human tumors thus may also contribute to increased malignancy. Site-directed mutagenesis of recombinant osteopontin protein indicates that an intact RGD sequence is required for cell adhesion and induction of chemotaxis, consistent with the idea that integrin-mediated signal transduction is a consequence of osteopontin binding to cells. Osteopontin may contribute to malignancy by inducing responses in host and/or tumor cells.

3T3 Cells↗
PubMed1995

Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions.

Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins ("delRGD" or "RGE") supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions.

3T3 Cells↗
PubMed1995

Effects of the disintegrin eristostatin on individual steps of hematogenous metastasis.

Adhesion molecules, including integrins, are important for interactions of cancer cells with vessel walls, a step leading to cancer metastasis. Disintegrins block the action of integrins by binding to them. We tested the hypothesis that the disintegrin eristostatin would block metastasis by interfering with cancer cell adhesion to vessel walls, thus preventing extravasation. Experimental metastasis assays, in which B16F1 melanoma cells (controls vs eristostatin-treated, 25 micrograms/ml) were injected via mesenteric veins of anesthetized C57BL/6 mice, showed that eristostatin reduced (P < 0.05) the mean number of liver metastases from 14.4 to 0.6 at 11 days postinjection (p.i.). We examined three different steps in metastasis at which eristostatin could have exerted its effect, namely, cell arrest, extravasation, and migration. Control and eristostatin-treated B16F1 cells were fluorescently labeled and examined by videomicroscopy in liver microcirculation in vivo at various times up to 14 days p.i. Measurements of vessel size in which cell arrest occurred and length/width ratio of arrested cells showed only small differences between control and eristostatin-treated cells. Eristostatin treatment did not prevent extravasation, and the timing and process of extravasation were similar for both treated and control cells; by 3-4 days p.i. more than 90% of the cells had extravasated or were in the process. Eristostatin also did not affect the ability of extravasated cells to migrate through the extracellular matrix to the subcapsular region where tumors later form. Therefore, we conclude that eristostatin exerted its primary effect by regulating the number of individual cancer cells that grow after extravasation.

Animals↗
PubMed1995

Steps in tumor metastasis: new concepts from intravital videomicroscopy.

Metastases are responsible for the majority of failures in cancer treatment. Clarifying steps in metastasis and their molecular mechanisms will be important for the development of anti-metastasis therapeutic strategies. Considerable progress has been made in identifying molecules involved in metastasis. However, because of the nature of assays that have been available, conclusions about steps in metastasis and their molecular bases have been drawn primarily from inference. In order to complete the picture of how metastases form, a technique is needed to directly watch the process in vivo as it occurs over time. We have developed an intravital videomicroscopy (IVVM) procedure to make such observations possible. Results from IVVM are providing us with new conceptual understanding of the metastatic process, as well as the nature and timing of the contributions of molecules implicated in metastasis (e.g. adhesion molecules and proteinases). Our findings suggest that early steps in metastasis, including hemodynamic destruction and extravasation, may contribute less to metastatic inefficiency than previously believed. Instead, our results suggest that the control of post-extravasation growth of individual cancer cells is a significant contributor to metastatic inefficiency. Thus, this stage may be an appropriate target for design of novel strategies to prevent metastases.

Animals↗
PubMed1995

Effect of tocotrienols on the growth of a human breast cancer cell line in culture.

The tocotrienol-rich fraction (TRF) of palm oil consists of tocotrienols and some alpha-tocopherol (alpha-T). Tocotrienols are a form of vitamin E having an unsaturated side-chain, rather than the saturated side-chain of the more common tocopherols. Because palm oil has been shown not to promote chemically-induced mammary carcinogenesis, we tested effects of TRF and alpha-T on the proliferation, growth, and plating efficiency (PE) of the MDA-MB-435 estrogen-receptor-negative human breast cancer cells. TRF inhibited the proliferation of these cells with a concentration required to inhibit cell proliferation by 50% of 180 microgram/mL whereas alpha-T had no effect at concentrations up to 1000 microgram/mL as measured by incorporation of [3H]thymidine. The effects of TRF and alpha-T also were tested in longer-term growth experiments, using concentrations of 180 and 500 microgram/mL. We found that TRF inhibited the growth of these cells by 50%, whereas alpha-T did not. Their effect on the ability of these cells to form colonies also was studied, and it was found that TRF inhibited PE, whereas alpha T had no effect. These results suggest that the inhibition is due to the presence of tocotrienols in TRF rather than alpha T.

Antioxidants↗
PubMed1995

O6-methylguanine-DNA methyltransferase in tumors and cells of the oligodendrocyte lineage.

BACKGROUND: Oligodendrogliomas respond to nitrosourea-based chemotherapy and are induced in rats following transplacental exposure to ethylnitrosourea, observations suggesting that neoplastic and normal cells of the oligodendrocyte lineage are "sensitive" to nitrosoureas. Nitrosoureas alkylate DNA at O6-guanine with repair mediated by O6-methylguanine-DNA methyltransferase (MGMT). The cytotoxic and carcinogenic properties of the nitrosoureas appear related to MGMT activity. METHODS: To explore why oligodendrogliomas respond to chemotherapy, we measured MGMT activity in five chemosensitive human oligodendrogliomas and in rat oligodendrocyte lineage cells. We also measured MGMT activity in rat astrocytes and compared the cytotoxic effects of carmustine (BCNU) on oligodendrocyte lineage cells and astrocytes. RESULTS: Low levels of MGMT activity were found in five of five human oligodendrogliomas. Cultures of neonatal rat glia enriched for oligodendrocyte lineage cells also had low levels of MGMT activity, approximately one-third that found in astrocytes (p < 0.02), and oligodendrocyte lineage cells were more sensitive to BCNU than astrocytes. CONCLUSIONS: Low MGMT activity may contribute to the chemosensitivity of some human oligodendrogliomas and rat oligodendrocyte lineage cells also have low levels. If drug resistance mechanisms in tumors reflect the biochemical properties of their cells of origin, then normal glia may serve as a laboratory substitute for human glioma.

Adult↗
PubMed1995

Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell.

The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the chain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha v beta 3 integrin-binding secreted phosphoprotein implicated in a number of developmental, physiological, and pathological processes. We report that in T24 H-ras-transformed NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a novel ras-activated enhancer, distinct from known ras response elements, that appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assays, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was generated at elevated levels by cell lines that are metastatic. Southwestern blotting and UV light cross-linking studies indicated the presence of several proteins able to interact with this sequence. The proteins that form these complexes have molecular masses estimated at approximately 16, 28, 32, 45, 80, and 100 kDa. Because the approximately 16-kDa protein was responsible for complex A formation, we have designated it MATF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be similarly controlled by MATF.

3T3 Cells↗
PubMed1995

Osteopontin and beta 3 integrin are coordinately expressed in regenerating endothelium in vivo and stimulate Arg-Gly-Asp-dependent endothelial migration in vitro.

Osteopontin is an Arg-Gly-Asp (RGD)-containing acidic glycoprotein postulated to mediate cellular adhesion and migration in a growing number of normal and pathological conditions through interaction with integrin molecules. In this report, we have investigated the potential contributions of osteopontin and one of its receptors, the alpha v beta 3 integrin, to endothelial regenerative processes by using both in vivo and in vitro models. In vivo, uninjured rat arterial endothelium had undetectable levels of osteopontin and beta 3-integrin mRNA by in situ hybridization. After balloon catheter denudation, osteopontin mRNA levels correlated temporally and spatially with active endothelial proliferation and migration, with the highest levels observed at the wound edge between 8 hours and 2 weeks after injury, declining to uninjured levels at 6 weeks, when regeneration was complete. Osteopontin protein levels, as determined by immunocytochemistry, paralleled the time course of mRNA expression. Likewise, beta 3-integrin mRNA and protein levels were substantially elevated in regenerating endothelial cells but were not detectable in uninjured or healed endothelium. In vitro, rat smooth muscle cell-derived and bacterial expressed mouse recombinant osteopontins both stimulated the adhesion and directed migration of bovine aortic endothelial cells through interactions with the alpha v beta 3 receptor. Structural mutants of osteopontin confirmed the importance of the RGD domain for both adhesion and migration of endothelial cells through alpha v beta 3. These data suggest important roles for osteopontin and beta 3 integrin in regenerating endothelium.

Animals↗
PubMed1994

Inhibition of Arg-Gly-Asp (RGD)-mediated cell adhesion to osteopontin by a monoclonal antibody against osteopontin.

Osteopontin (OPN), a secreted phosphoprotein, has been implicated in various biological phenomena (e.g. bone development, sepsis, tumor progression, and metastasis). Its role in any context is poorly understood. OPN contains a conserved Gly-Arg-Gly-Asp-Ser (GRGDS) sequence, and binds to cells via integrin-mediated mechanisms. Using recombinant human osteopontin-glutathione S-transferase fusion protein and our improved hybridoma fusion partner (Sp2/mIL6), we raised murine monoclonal antibodies against osteopontin. We characterized two antibodies that recognize not only recombinant but also native human osteopontin. These antibodies do not cross-react with mouse osteopontin (recombinant protein or that secreted by ras-transformed NIH 3T3 cells), or bovine bone osteopontin, suggesting that they recognize epitopes unique to human OPN. One antibody specifically inhibited adhesion of MDA-MB-435 human breast cancer cells and ras-transformed NIH 3T3 cells to human osteopontin. This antibody failed to recognize osteopontin cleaved by thrombin, which cleaves adjacent to the cell binding domain. We previously showed that thrombin cleavage reduces osteopontin cell binding activity. Thus we postulate that this monoclonal antibody recognizes and interferes with the function of the RGD/thrombin cleavage region of human OPN.

3T3 Cells↗
PubMed1994

Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth.

It is widely accepted that a major role of matrix metalloproteinases in the metastatic process is degradation of basement membrane during cancer cell invasion. We tested the hypothesis that the reduction in metastatic potential which has been demonstrated for B16F10 melanoma cells genetically engineered to overexpress tissue inhibitor of metalloproteinase-1 (TIMP-1) is caused by a decrease in their ability to extravasate. Using intravital videomicroscopy of chick embryo chorioallantoic membrane, we studied extravasation of B16F10 cells and B16F10 cells transfected to overexpress TIMP-1. More than 800 cells in 36 chick embryos were analyzed for each cell line during 72 h postinjection. TIMP-1 upregulation had no effect on the time course of extravasation, virtually all cells from both cell lines having extravasated by 36 h. We also studied the morphology of micrometastases at days 3 and 7. Lack of contact between cancer cells within micrometastases at day 3 and reduction in size and number of tumors at day 7 were observed for TIMP-1 overexpressor cells compared to B16F10. Our findings illustrate that the imbalance between TIMP and metalloproteinases created by overexpression of TIMP-1 in B16F10 cells reduces their metastatic ability in vivo by affecting tumor growth postextravasation.

Animals↗