Platelet-derived growth factor-B increases colon cancer cell growth in vivo by a paracrine effect

PDGF-B released from colon tumor cells regulated tumor growth in athymic mice in a paracrine manner by inducing blood vessel formation. A positive correlation was found between expression of PDGF B-chain in cells grown in vitro and the number of factor VIII-positive blood vessels in tumors induced by three classes of colon carcinoma cell lines. Elevated expression of PDGF-B was also correlated with tumor size. Each cell line had the same mutations in the colon cancer genes APC, DCC, and p53 and had wild type c-K-ras genes (Huang et al. [1994] Oncogene, 9:3701-3706.) eliminating the possibility that any differences in tumor blood vessel formation were due to mutations and/or deletions in these genes. Colon carcinoma cells released biologically active PDGF capable of stimulating the growth of NIH3T3 cells, which was inhibited by neutralizing antisera to PDGF-AB chains. An inverse correlation was found between induction of factor VIII-positive blood vessels and expression of vascular endothelial growth factor (VEGF), while no correlation was seen with expression of either TGF alpha or k-FGF. Basic fibroblast growth factor (FGF) expression was not detected in these tumor cells. TGF beta 1 was capable of inducing PDGF-B expression in the undifferentiated U9 colon carcinoma cell line, but this sensitivity was not seen in differentiated cells. In contrast, TGF beta 1 inhibited VEGF expression in both undifferentiated cells and differentiated colon cancer cells. Thus, TGF beta 1 has two roles in the growth of undifferentiated U9 colon carcinoma cells in vivo: direct stimulation of cell proliferation as we have showed in earlier studies, and an increase in angiogenesis by inducing PDGF-B.     

Growth inhibitory effects of aromatic fatty acids on ovarian tumor cell lines

Epithelial ovarian cancer is a major cause of cancer-related mortality in women, making the search for new treatment modalities essential. Sodium phenylacetate (NaPa), a phenylalanine derivative, has been shown to induce cytostasis and differentiation by inhibiting protein isoprenylation. Similar effects have been observed with phenylbutyrate, a phenylacetate congener. We examined in parallel the growth inhibitory activity against human ovarian carcinoma cell lines of phenylacetate, phenylbutyric acid (PB), and certain related compounds, and comparisons were made with lovastatin. On a molar basis, hydroxykynurenine and kynurenine showed the highest activity followed by PB and NaPa. Ovarian carcinoma cell lines were also sensitive to lovastatin in micromolar concentrations. Additive effects were observed when PB was combined with cisplatin or when NaPa or PB were combined with lovastatin. NaPa and PB, but not kynurenine, inhibited incorporation of [3H]mevalonate into ovarian carcinoma cells. An immune modulatory role might also be suggested for PB because it resulted in increased ovarian tumor cell expression of human leukocyte antigen class I and the cluster of differentiation molecule CD58, whereas transforming growth factor-beta2 expression was decreased. Phenylbutyrate, which is the ester form of PB, has shown acceptable pharmacological properties and clinical responses in patients with other malignancies, and might be considered for evaluation in ovarian cancer.     

IFN-gamma induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression

The mechanism by which IFN-gamma inhibits tumor cell growth has not been fully understood. Here we report that IFN-gamma up-regulated the expression of Fas and Fas ligand (FasL) on HT29 cells, a human colon adenocarcinoma cell line, and subsequently induced apoptosis of these cells. The kinetics of cell death in IFN-gamma-treated HT29 cells paralleled the increase in the levels of Fas and FasL expression. We further show that IFN-gamma up-regulated the expression of Fas and FasL in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. Correspondingly, IFN-gamma induced cell death in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. IFN-gamma-induced cell death was inhibited by caspase-1 inhibitors. Our results suggest that cell growth inhibition by IFN-gamma is due to apoptosis mediated by Fas and FasL interaction.     

Enterocytic differentiation correlates with changes in the fine structure and sulfation of perlecan in HT29 human colon carcinoma cells

Undifferentiated HT29 and differentiated HT29G-human colon carcinoma cells have been used to study the changes in proteoglycan production and structure associated with enterocytic cell differentiation. Differentiated cells incorporate twice as much sulfate than undifferentiated cells when labeled with [35S]sulfate. Both cell lines produce a heparan sulfate proteoglycan which was purified by ion-exchange. The heparan sulfate proteoglycan from differentiated HT29G- cells is larger and more homogeneous in size than that produced by undifferentiated HT29 cells. No differences in the core protein structure were observed. The detailed structural analysis of the heparan sulfate chains revealed that the structure of these chains follows the standard rules for these glycosaminoglycans with N-sulfated domains and N-acetylated domains. The main finding was that differentiated HT29G- cells have a degree of higher sulfation than HT29 cells. These differences were found to affect primarily 6-O-sulfated positions.     

Protease activation and cleavage of poly(ADP-ribose) polymerase: an integral part of apoptosis in response to photodynamic treatment

BACKGROUND & AIMS: We tested the hypothesis that short-chain organic acids in the colon derived from dietary pectin, wheat bran, and oat bran are protective against the development of colon cancer because they induce transforming growth factor (TGF)-beta1, which in turn inhibits cell growth by inducing cyclin-directed kinase (cdk) inhibitors. METHODS: U4 human colon carcinoma cells differentiate into water- and salt-transporting columnar enterocytes and therefore model normal colonocytes. The composition and kinase activity of cdk/cyclin complexes were determined by immunoprecipitation and Western blotting studies in U4 cells treated in vitro with short-chain fatty acid (SCFA) mixtures that mimic the digestion products of wheat bran, oat bran, pectin, and cellulose (as control), which is largely unfermentable. RESULTS: Induction of the cdk inhibitors p21cip1 and p27kip1 by fiber-mimicking SCFA mixtures occurs much more rapidly and is many-fold greater than their induction by TGF-beta1. The SCFA mixtures most effective in causing growth inhibition and cdk inhibitor production mimicked those from wheat bran > oat bran > pectin. CONCLUSIONS: cdk inhibitor induction by SCFA mixtures is not mediated by TGF-beta1. The SCFA mixture mimicking digested wheat bran fiber was the most effective of all mixtures tested in inhibiting cell growth through induction of cdk inhibitors.     

Expression and posttranslational fate of cathepsin D in HT-29 tumor cells depend on their enterocytic differentiation state

In the present work, we analyzed the variations in the expression and trafficking of cathepsin D (CD), a lysosomal endopeptidase, associated with the enterocytic differentiation of the human colon carcinoma HT-29 cell line. In spite of the fact that the abundance of CD mRNA was severalfold higher in undifferentiated HT-29 cells than in their enterocyte-like differentiated counterparts, the intracellular levels of CD activity and protein were found to be much higher in the latter. The kinetic of transport of newly synthesized proCD was different in the two cell populations: (a) full conversion of proCD into the lysosomal mature form required more than 24 h in differentiated cells, whereas it was almost complete within 8 h in undifferentiated HT-29 cells; and (b) the extracellular release of proCD was shown to occur more rapidly and to a higher degree in undifferentiated than in differentiated cells. Most of the secreted proCD contained phosphomannoses. Secretion of beta-hexosaminidase activity doubled, whereas that of CD activity was unchanged, upon vacuolar alkalinization with ammonium chloride or chloroquine. Inhibition of the lysosomal-autophagic degradative pathway resulted in the accumulation of proCD molecules in undifferentiated HT-29 cells. Altogether these data suggest that: (a) the expression and the posttranslational fate of CD in HT-29 colon cancer cells are largely affected by the state of their enterocytic differentiation; and (b) in this cell line the acid-dependent mannose 6-phosphate receptor pathway is, at best, little involved in the trafficking of CD.     

Comparative effects of overexpression of p27Kip1 and p21Cip1/Waf1 on growth and differentiation in human colon carcinoma cells

Recent studies have shown that decreased expression of p27Kip1 is associated with high grade tumors and an unfavorable prognosis in several types of human cancer. To clarify the role of p27Kip1 in colon cancer, we have overexpressed this protein in the HT29 colon cancer cell line. The derivatives displayed an increase in the p27Kip1 protein in cyclin E/CDK2 immunoprecipitates and a decrease in cyclin E-associated kinase activity when compared to vector control clones, providing evidence that the overexpressed protein was functional. Clones with a high level of p27Kip1 displayed partial growth inhibition in monolayer culture and a decrease in plating efficiency, even though they expressed increased levels of the cyclin D1 protein. Using alkaline phosphatase expression as a marker, we found that the p27Kip1 overexpressor clones displayed a 2-3-fold increase in sensitivity to induction of differentiation by 2 mM sodium butyrate. In contrast to these results, derivatives of HT29 cells that stably overexpressed p21Cip1/Waf1 displayed decreased sensitivity to the induction of differentiation. These findings may explain why decreased levels of p27Kip1 in certain human cancers is associated with high grade (poorly differentiated) tumors, and suggest that strategies that increase the level of p27Kip1 may be useful in cancer therapy.     

19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.     

Enhanced antitumor efficacy of cisplatin in combination with ALRT1057 (9-cis retinoic acid) in human oral squamous carcinoma xenografts in nude mice

Cisplatin (DDP) is commonly used to treat head and neck tumors. Therapy frequently fails due to development of DDP resistance or toxicities associated with DDP therapy. In this study, effects of ALRT1057 [9-cis retinoic acid (9-cis RA)] on DDP cytotoxicity were studied in a human oral squamous carcinoma xenograft model. Mice bearing xenografts were dosed p.o. daily 5 days/week with 30 mg/kg 9-cis RA and/or i.p. twice weekly with 0.3-0.9 mg/kg DDP. Maximum tolerated doses of 9-cis RA and DDP were approximately 60 and >/=2.9 mg/kg, respectively, under their dosing schedules and routes of administration. Control tumors grew rapidly with mean doubling times of 4 +/- 1 days and reached mean volumes of 1982 +/- 199 (SE) mm3 after 24 days. DDP at doses of 0.3, 0.45, and 0.9 mg/kg inhibited tumor growth by 28, 47, and 86%, respectively, 24 days after tumor cell implantation. Thirty mg/kg 9-cis RA inhibited tumor growth by 25%. In combination, 0.3 mg/kg DDP + 30 mg/kg 9-cis RA inhibited tumor growth by 68%; 0.45 mg/kg DDP + 30 mg/kg 9-cis RA inhibited growth by 78%. These decreases were greater than those that would have been produced by either agent summed separately. Of importance, at doses of 9-cis RA that enhanced DDP cytotoxicity, no change in dose tolerance was observed as compared to tolerances observed for either agent alone, indicating that 9-cis RA increased sensitivity to DDP without altering systemic toxicity. In addition, 9-cis RA profoundly altered squamous cell carcinoma phenotypes by suppressing squamous cell differentiation, resulting in tumors with increased numbers of basal cells. In contrast, DDP selectively depleted proliferating basal cells from carcinomas. In combination, morphological changes produced by 9-cis RA alone predominated, suggesting a possible basis for enhanced DDP sensitivity in tumors exposed to both agents. These data demonstrate that 9-cis RA enhances tumor sensitivity to DDP, and suggest that this combination should be tested in Phase I-II clinical trials for its potential for improving anticancer therapy of squamous cell cancers.     

Differential effects of deoxycholic acid on proliferation of neoplastic and differentiated colonocytes in vitro

The secondary bile acid deoxycholic acid is believed to be a promoter of large bowel cancer, in part by inducing colonic epithelial proliferation. The effects of deoxycholic acid on [3H]thymidine incorporation by the human colon cancer cell line HT29 and two differentiated subclones were measured and compared. The subclone HT29-C1 has features of mature absorptive cells and HT29-N2 cells secrete mucus under cholinergic control. The three cell lines were treated with deoxycholic acid (DCA) at concentrations of 0, 5, 10, 50, 100, 150, and 300 microM for 3, 6, 9, 15, 24, and 48 hr. A significant increase in proliferation was noted in HT29 cells only at 6 hr with 5 and 10 microM deoxycholic acid. Neither the subclone HT29-C1, nor HT29-N2 cells exhibited significant change in [3H]thymidine incorporation with DCA at these concentrations or time points. Higher doses of deoxycholic acid above 50 microM and duration of exposure greater than 24 hr were cytotoxic to all three cell lines. The proliferative effects of DCA in HT29 cells were not paralleled by changes in protein kinase C activity or protein kinase C isoform expression. Quantitative and qualitative differences in PKC isoform expression were not noted in the three cell lines used in this study. The proliferative effects of DCA on HT29 cells appear to be independent of the PKC signal transduction pathway.     

Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29-D4 human colonic carcinoma cells

To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa beta-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two alphabeta heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the alpha-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR beta-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into alphabeta heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, approximately 1.5 nM), but did not bind insulin significantly; the latter interacted with the native IR only (Kd, approximately 4 nM). In the differentiated HT29-D4 cell monolayer, all receptor species were strongly polarized (>97%) toward the basolateral membrane. Moreover, HT29-D4 cell differentiation was accompanied by an approximately 2-fold increase in the number of IR, whereas the number of IGF-I-binding sites was unaltered. However, in differentiated HT29-D4 cells, approximately 55% of the latter were involved in HR vs. approximately 20% in undifferentiated HT29-D4 cells. Thus, HT29-D4 cell differentiation is characterized by an up-regulation (approximately 3-fold) of the level of HR coupled to a down-regulation (approximately 40%) of the level of native tetrameric IGF-IR. Alterations were induced early during the cell differentiation process, i.e. 5 days postconfluence, and remained unchanged for at least 21 days. Taken together, these results suggest that the IGF-II autocrine loop in HT29-D4 cells may trigger distinct signaling pathways if it activates native IGF-IR, which predominate in undifferentiated cells, or if it activates HR, which are up-regulated in differentiated cells.     

Chemotherapy-induced mobilization of karyotypically normal PBSC for autografting in CML

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O-sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.     

Growth, morphology and chemosensitivity studies on postconfluent cells cultured in 'V'-bottomed microtiter plates

This study assessed the growth pattern, cellular organisation and chemosensitivity of established human tumour cell lines growing as postconfluent cultures in 'V'-bottomed, 96-well microtiter plates. Cross-sections of the colon (HT29, SW620, SW1116), ovarian (A2780) and head and neck (UM-SCC-22B) carcinoma microcultures allowed in situ evaluation of the cellular organisation in the wells. After 5 days of growth, every cell line had reached confluence, but each of them displayed a specific pattern of cell stacking which ranged from two to ten layers. Postconfluent HT29 cells displayed morphologic features suggestive of some degree of enterocytic differentiation. Growth and cytotoxicity could be studied reliably and reproducibly in this system with the sulforhodamine B protein assay. Against HT29 postconfluent cultures, the EC50's (drug concentrations producing absorbance readings 50% lower than those of non-treated wells) of 5-fluorouracil and of the ether lipid, hexadecylphosphocholine, were 1 mM and 50 microM respectively. The possibility to perform chemosensitivity tests using semiautomated microtiter plate technology supports further evaluation of this system as an alternative antitumour drug testing model.     

1,25-Dihydroxyvitamin D3 receptor as a marker of human colon carcinoma cell line differentiation and growth inhibition

The antiproliferative action of 1,25-dihydroxyvitamin D3 in osteosarcoma, breast carcinoma, and colon carcinoma cell lines has been described. In this study, the level of vitamin D receptor was analyzed in a panel of colon adenoma and adenocarcinoma cell lines and the receptor level was correlated with the response to treatment with 1,25-dihydroxyvitamin D3. Ribonuclease protection and ligand-binding assays quantitated the level of vitamin D receptor mRNA expression and the level of functional receptors, respectively. The more well-differentiated cell lines, such as VACO 330, showed higher levels of vitamin D receptor than less-differentiated cell lines, such as SW620. Proliferation assay, clonogenic assay, and growth curve study in HT29 and SW620 cell lines assessed the antiproliferative effect of 1,25-dihydroxyvitamin D3 at concentrations ranging from 10(-11) to 10(-6) M. HT29 showed significant (P < 0.05) growth inhibition at 10(-9) to 10(-6) M concentrations, but growth of SW620 remained unchanged. The amount of vitamin D receptor in 12 malignant colonic tumors was compared with that of adjacent normal tissue, and in 9 cases, the tumor expressed a lower vitamin D receptor level. Our results suggest that the level of vitamin D receptor correlates with the degree of differentiation in human colon cancer cell lines and may serve as a useful biological marker in predicting clinical outcome in patients.     

Differential coupling of alpha1-, alpha2-, and beta-adrenergic receptors to mitogen-activated protein kinase pathways and differentiation in transfected PC12 cells

Three adrenergic receptor families that selectively activate three different G proteins (alpha1/Gq/11, alpha2/Gi, and beta/Gs) were used to study mitogen-activated protein kinase (MAPK) activation and differentiation in PC12 cells. PC12 cells were stably transfected with alpha1A-, alpha2A-, or beta1-adrenergic receptors (ARs) in an inducible expression vector, and subclones were characterized. Norepinephrine stimulated inositol phosphate formation in alpha1A-transfected cells, inhibited cyclic adenosine 3'5'-monophosphate (cAMP) formation in alpha2A-transfected cells, and stimulated cAMP formation in beta1-transfected cells. Nerve growth factor activated extracellular signal-regulated kinases (ERKs) in all cell lines; however, norepinephrine activated ERKs only in alpha1A- and beta1-transfected cells but not in alpha2A-transfected cells. Norepinephrine also activated c-Jun NH2-terminal kinase and p38 MAPK in alpha1A-transfected cells but not in beta1- or alpha2A-transfected cells. Norepinephrine caused differentiation of PC12 cells expressing alpha1A-ARs but not those expressing beta1- or alpha2A-ARs. However, norepinephrine acted synergistically with nerve growth factor in promoting differentiation of cells expressing beta1-ARs. Whereas ERKs are activated by Gi- but not Gs-linked receptors in many fibroblastic cell lines, we observed the opposite in PC12 cells. The results show that activation of the different G protein signaling pathways has different effects on MAPKs and differentiation in PC12 cells, with Gq signaling pathways activating all three major MAPK pathways.     

Protein kinase C-dependent tyrosine phosphorylation of p130cas in differentiating neuroblastoma cells

The cell signaling docking protein p130cas became tyrosine-phosphorylated in SH-SY5Y human neuroblastoma cells during induced differentiation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum or a combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The differentiating cells develop a neuronal phenotype with neurites and growth cones and sustained activation of protein kinase C (PKC) and pp60c-src. The TPA-induced p130cas phosphorylation increased within 5 min of stimulation and persisted for at least 4 days, whereas bFGF/IGF-I-induced p130cas phosphorylation was biphasic. However, the increase in tyrosine phosphorylation of p130cas was not restricted to differentiation inducing stimuli. The phosphorylation was blocked by the specific PKC inhibitor GF 109203X, and transient transfection with active PKC-epsilon induced p130cas tyrosine phosphorylation. pp60c-src, known to directly phosphorylate p130cas in other cell systems, was not activated after stimulation with TPA or bFGF/IGF-I for up to 30 min, and the initial p130cas phosphorylation was resistant to the Src family kinase inhibitor herbimycin A. However, in long term stimulated cells, herbimycin A blocked the induced phosphorylation of p130cas. Also, overexpression of src induced phosphorylation of p130cas. p130cas protein and phosphorylated p130cas were present in growth cones isolated from differentiated SH-SY5Y cells. Inhibition of PKC activity in differentiating cells with GF 109203X leads to a rapid retraction of growth cone filopodia, and p130cas phosphorylation decreased transiently (within minutes). Growth cones isolated from these cells were virtually devoid of phosphorylated p130cas. These data suggest a function for p130cas as a PKC downstream target in SH-SY5Y cells and possibly also in their growth cones.     

Autocrine transforming growth factor alpha provides a growth advantage to malignant cells by facilitating re-entry into the cell cycle from suboptimal growth states

CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-alpha transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-alpha-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-alpha-transfected cells indicate that autocrine TGF-alpha functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-alpha. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-alpha.     

结直肠癌中NF-κB表达的研究

Objective: The aim of the study was to explore the protein level of NF-κB change in carcinoma and different grades of tumor cells differentiation tissue in colorectal cancer patients. Methods: This was a comparative study between normal and carcinoma tissues and in different grades of tumor cells differentiation tissue in colorectal cancer patients. Expression of NF-κΒ were assessed by immunohistochemical method using rabbit polyclonal antibodies against human p65 NF-κΒ proteins. Results: There was none or very little expression of NF-κB in non-neoplastic colon epithelial cells, while the expression of it's protein was significantly increased (P < 0.01) in adjacent cancerous cells. Moreover, there was a significant increase in the mean expression of NF-κB-p65 between poorly differentiated malignant epithelial cell and well-differentiated cells (P < 0.05). Conclusion: NF-κB-p65 may play an essential role in colorectal carcinogenesis and may be valuable for diagnosis, evaluating malignancy extent and prognosis.     

Impact of the putative differentiating agents sodium phenylbutyrate and sodium phenylacetate on proliferation, differentiation, and apoptosis of primary neoplastic myeloid cells

Sodium phenylacetate (PA) and sodium phenylbutyrate (PB) are aromatic fatty acids that can effect differentiation in a variety of cell lines at doses that may be clinically attainable. We have studied the impact of these two agents on lineage- and differentiation stage-specific antigen expression, proliferation, apoptosis, and clonogenic cell survival in primary cultures of bone marrow samples from patients with myeloid neoplasms at presentation and in remission and from normal volunteers. PB inhibited the proliferation of primary acute myeloid leukemia cells in suspension culture with an ID50 of 6.6 mM, similar to its ED50 in cell lines. At higher doses (>/=5 mM), PB also induced apoptosis. PB inhibited clonogenic leukemia cell growth with a median ID50 of less than 2 mM; however, colony-forming units-granulocyte/macrophage from patients with myelodysplasia and normal volunteers were inhibited with a similar ID50. In contrast to PB, its metabolite PA had no significant effect on either acute myeloid leukemia proliferation or apoptosis. Expression of the monocytic marker CD14 was increased in monocytic and myelomonocytic leukemias in response to PB, and to a lesser extent, PA. Surprisingly, both agents appeared to increase expression of the progenitor cell antigen CD34, as well as the DR locus of the human leukocyte antigen. These data indicate that PB, but not its metabolite PA, has significant cytostatic and differentiating activity against primary neoplastic myeloid cells at doses that may be achievable clinically.