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Crude trypsin is the agent of choice for the disaggregation of human tonsil. A great variability which is not related to specific activity exists among lots of crude trypsin. An efficient lot of crude trypsin is necessary to obtain optimal proportions of tonsillar cells for the purification of lymphocytes and plasma cells by a previously reported technique. [ABSTRACT FROM AUTHOR]

The phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway is a central regulator in cell proliferation, growth, and angiogenesis. Inhibition of this pathway therefore is a major strategy for cancer chemotherapy. In order to induce the maximal therapeutic outcome in cancer treatment, vertical inhibition of the PI3K/AKT/mTOR pathway or horizontal inhibition of PI3K/AKT/mTOR and other kinases has been reported. In this review, we discuss the drug design and clinical development of dual inhibitors of PI3K and mTOR as well as the mTOR-selective inhibitors, classified based on the mechanism of action and the chemical structures. Structural determinants for increasing selectivity toward PI3Kα or mTOR are revealed from the structure-activity relationship of the reported inhibitors. Current clinical development in combination therapy of inhibitors involving in the PI3K/AKT/mTOR pathway is also discussed.

In this study, we address whether TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGFbeta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGFbeta. MCF7L TGFbetaRII-transfected cells, which have autocrine TGFbeta activity, were more sensitive to EB1089 than MCF7L cells. TGFbeta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGFbeta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGFbeta isoforms and/or TGFbeta receptors was induced by the analogs in the vitamin D3 and TGFbeta sensitive cells. Vitamin D3 analogs did not induce TGFbeta or TGFbeta receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGFbeta activity through increasing expression of TGFbeta isoforms and/or TGFbeta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGFbeta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGFbeta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGFbeta/vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGFbeta and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGFbeta and vitamin D signaling is also PI 3-kinase pathway dependent., (Copyright 2001 Wiley-Liss, Inc.)

We have previously reported that Sp3 acts as a transcriptional repressor of transforming growth factor-beta receptors type I (RI) and type II (RII). We now present data suggesting that treatment of MCF-7L breast and GEO colon cancer cells with 5-aza cytidine (5-azaC) leads to down-regulation of Sp3 and the concomitant induction of RI and RII. Western blot and gel shift analyses on 5-azaC-treated MCF-7L and GEO nuclear extracts indicated reduced Sp3 protein levels and decreased binding of Sp3 protein to radiolabeled consensus Sp1 oligonucleotide. Southwestern analysis detected decreased binding of Sp3 to RI and RII promoters in 5-azaC-treated MCF-7L and GEO cells, suggesting a correlation between decreased Sp3 binding and enhanced RI and RII expression in these cells. Reverse transcription-polymerase chain reaction and nuclear run-on data from 5-azaC-treated MCF-7L and GEO cells indicated down-regulation of Sp3 mRNA as a result of decreased transcription of Sp3. We reported earlier that 5-azaC treatment induces RI and RII expression through increased Sp1 protein levels/activities in these cells. These studies demonstrate that the effect of 5-azaC involves a combination of effects on Sp1 and Sp3.

The pancreatic cancer cell line, MIA PaCa-2 is not responsive to transforming growth factor beta (TGF-beta) because of a lack of expression of the TGF-beta type II receptor (RII). We show that the lack of RII expression is caused by a deficit of the transcription factor Sp1. Nuclear run-off assays and Western immunoblot showed low levels of transcription and protein levels of Sp1, respectively. Treatment of MIA PaCa-2 cells with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine, resulted in an increase in the rate of Sp1 transcription, in Sp1 protein expression, and in the binding of Sp1 to the RII promoter. Ectopic expression of Sp1 cDNA in MIA PaCa-2 cells led to an increase in RII promoter-chloramphenicol acetyltransferase activity and RII expression. Expression of Sp1 cDNA also caused a reduction in both growth and clonogenicity that was associated with restoration of responsiveness to TGF-beta. Conversely, cells that express RII (BxPC-3 and MIA PaCa-2 Sp1 transfectants) when treated with mithramycin, an inhibitor of Sp1 binding, showed a reduction in RII mRNA expression. The reduction of RII mRNA was attributed to a decrease in RII promoter-chloramphenicol acetyltransferase activity that was associated with a decrease in Sp1 binding to the RII promoter. These data indicate that transcriptional repression of the Sp1 gene in MIA PaCa-2 cells plays a role in the transcriptional inactivation of the RII gene and thus lack of responsiveness to TGF-beta.

MCF-7E breast cancer cells express transforming growth factor-beta (TGF-beta) receptors RI and RII in comparison to MCF-7L cells. We present data showing that Sp3 acts as a transcriptional repressor of RI and RII in MCF-7L cells and GEO colon cancer cells. MCF-7L and GEO cells express high levels of Sp3 protein. Gel shift analysis indicated enhanced binding of Sp3 from MCF-7L cells to a consensus Sp1 oligonucleotide. Southwestern data indicated increased binding of Sp3 to RI and RII promoters in MCF-7L cells, suggesting a correlation between Sp3 binding and reduced expression of TGF-beta receptors in MCF-7L cells. Cotransfection of CMV-Sp3 cDNA with RI and RII promoter-luciferase reporter constructs decreased RI and RII promoter activities by 70% in MCF-7E and GEO cells. Southwestern analysis detected the binding of transiently expressed Sp3 to RI and RII promoters in MCF-7E cells. Significantly, ectopic Sp3 expression led to repression of RI and RII transcripts in MCF-7E cells. This report demonstrates that inappropriate overexpression of Sp3 is a mechanism that contributes to repression of TGF-beta receptors.

Akt, when activated by IGF/insulin, can phosphorylate forkhead transcription factors. We undertook this study to determine whether epidermal growth factor (EGF) treatment could produce a signaling cascade resulting in phosphorylation of the forkhead transcription factor FKHR in a breast cancer cell line, MDA-MB-231. After establishing ErbB1, cbl, PI3 kinase and Akt were activated in EGF treated MDA-MB-231, we determined by immunoblot with FKHR antiserum that the electrophoretic mobility of FKHR was retarded after EGF treatment. This mobility retardation was reversible by treatment with alkaline phosphatase, and immunoblot with phospho-Ser256 FKHR antibody further confirmed phosphorylation on an Akt consensus site after EGF treatment. EGF stimulated FKHR phosphorylation was blocked by the PI3 kinase inhibitor LY294002, and the ErbB1 inhibitor AG1478. FKHR immunoblotting after purification of nuclear and cytoplasmic proteins showed that EGF induced a simultaneous increase of FKHR in the cytoplasm and decrease in the nucleus. This finding was confirmed by immunofluorescence staining. Treatment of cells with pharmacological inhibitors of PI3 kinase or ErbB1 blocked this effect. Thus, these results demonstrate the phosphorylation and nuclear exclusion of FKHR after EGF treatment by a PI3 kinase dependent mechanism, and represent the first report of growth factor regulation of endogenous FKHR localization.

In this report, we describe the mechanism of TGF-beta receptor type I (RI) repression in the GEO human colon carcinoma cells. Treatment of GEO cells with the DNA methyltransferase inhibitor, 5 azacytidine induced RI expression and restored TGF-beta response. A stably transfected RI promoter-reporter construct (RI-Luc) expressed higher activity in the 5 aza C treated GEO cells, suggesting the activation of a transactivator for RI transcription. Gel shift analysis indicated enhanced binding of proteins from the 5 aza C treated nuclear extracts to radiolabeled Sp1 oligonucleotides specifically contained in the RI promoter. Protein stability studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5 aza C treated GEO cells. Further, transfection of Sp1 cDNA into untreated GEO control cells increased RI promoter activity and thus induced RI expression. 5 aza C mediated Sp1 expression in Sp1 deficient GEO colon and MCF-7 breast cancer cells also enhanced the activity of several other Sp1 dependent promoters such as TGF-beta receptor type II (RII), Cyclin A and p21/waf1/cip1. These results indicate that restoration of Sp1 in several different types of Sp1 deficient cells leads to enhanced activation of a wide range of Sp1 dependent promoters.

Transforming growth factor alpha (TGFalpha) is widely expressed in malignant as well as normal cells and is involved in regulating cell growth and differentiation. Although processing of TGFalpha has been extensively studied in normal cells, there is little information regarding TGFalpha cleavage in malignant cells. Therefore, we compared the processing of TGFalpha in two human colon carcinoma cell lines. We found that there was a defective cleavage pattern for the TGFalpha precursor resulting in retention of partially processed TGFalpha on the cell surface of both the HCT116a2alphaS3 and CBS4alphaS2 cell lines. This raised the possibility that signaling from the resulting defective cleavage species could differ from that of soluble TGFalpha. The membrane-associated TGFalpha induced higher phosphorylation of EGFR on the cell surface of adjacent cells than equivalent levels of mature TGFalpha. The interaction of membrane bound TGFalpha precursor with the EGFR caused a slower internalization of activated EGFR relative to the internalization of the soluble TGFalpha/EGFR complexes. In addition, the tethered TGFalpha was resistant to the ability of protein-tyrosine phosphatases (PTPs) to reduce EGFR tyrosine phosphorylation, also contributing to higher activation of EGFR. The enhanced activation of EGFR by the tethered form of TGFalpha was reflected by higher activation of Grb2, SHC and Erk downstream mediators of EGF receptor signaling. The higher activation of EGFR by membrane tethered TGFalpha indicates that defective TGFalpha processing provides a mechanism whereby malignant cells can obtain a growth advantage over normal cells.

Autocrine TGFalpha is an important growth effector in the transformed phenotype. Growth stimulation of some colon cancer cells as well as other types of cancer cells is effected by activation of the epidermal growth factor receptor. Importantly, this receptor activation leads to further stimulation of TGFalpha transcription and increased peptide synthesis. However, the molecular mechanism by which TGFalpha transcription is activated is poorly understood. In this paper, we describe the localization of a cis-sequence within the TGFalpha promoter which mediates this stimulation. This region contains parallel cis-acting elements which interact to regulate both basal and EGF-induced TGFalpha expression. The well differentiated colon carcinoma cell line designated FET was employed in these studies. It produces autocrine TGFalpha but requires exogenous EGF in the medium for optimal growth. Addition of EGF to FET cells maintained in the absence of EGF resulted in a 2 - 3-fold increase of both TGF promoter activity and endogenous TGFalpha mRNA at 4 h. This addition of EGF also stimulated protein synthesis. The use of deletion constructs of the TGFalpha promoter in chimeras with chloramphenicol acetyl transferase localized EGF-responsiveness to between -247 and -201 within the TGFalpha promoter. A 25 bp sequence within this region conferred EGF-responsiveness to heterologous promoter constructs. Further use of deletion/mutation chimeric constructs revealed the presence of at least two interacting cis-elements, one binding a repressor activity and the other, an activator. Gel shift studies indicate the presence of distinct complexes representing activator and repressor binding, which are positively modulated by EGF. The type and amount of complexes formed by these proteins interact to regulate both the basal activity and EGF-responsiveness of the TGFalpha promoter. The interaction of an activator protein with an EGF-responsive repressor may serve to regulate the level of this progression-associated, transforming protein within tight limits.

Transforming growth factor betas (TGF-betas) are a growth factor family with negative autocrine growth functions for most epithelial cells including colon carcinoma cell lines. Both type I (RI) and type II (RII) transmembrane TGF-beta receptors have been shown to be indispensable for TGF-beta-mediated cell growth regulation. Previous studies using different model systems have shown that both overexpression of TGF-beta1 and transfection of antisense TGF-beta1 to reduce TGF-beta1 expression could lead to increased tumorigenicity. These results are seemingly contradictory and suggest that effects of TGF-beta modulation on malignant properties of cancer cells may be contextual. This study addresses this issue using human colon carcinoma cells (CBS and FET) to determine the effects of modulation of the various components of the TGF-beta system on in vitro and in vivo growth properties in two independent isogenic models of colon carcinoma. Cells were stably transfected with a tetracycline-repressible RII expression vector (CBS4-RII), a tetracycline-repressible expression vector containing a truncated RII cDNA lacking the serine/threonine kinase domain (CBS4-deltaRII and FET6-deltaRII), or with a vector containing the TGF-beta1 cDNA (CBS4-beta1S and FET-beta1S). Expression of the truncated RII reduced TGF-beta sensitivity, whereas overexpression of RII increased TGF-beta sensitivity. TGF-beta overexpression did not affect TGF-beta response. In vivo tumorigenicity assays revealed that CBS4-RII cells had lower tumorigenicity than control cells, whereas CBS4-deltaRII and CBS4-beta1S had higher tumorigenicity than controls. The CBS4 cells are poorly tumorigenic in athymic mice, and the wild-type FET6 cells are nontumorigenic. FET6-deltaRII cells formed rapidly growing tumors, and FET-beta1S cells also formed tumors. These data illustrate the paradoxical tumor-promoting and -suppressing effects of TGF-beta signaling activity in two isogenic model systems from human colon carcinomas, thus demonstrating that the effects of modulation of TGF-beta expression or TGF-beta signaling capability affects malignancy in a contextual manner.

We previously demonstrated that mutational inactivation of transforming growth factor beta type II receptors (RIIs) is very common among the 13% of human colon cancers with microsatellite instability. These mutations principally cluster in the BAT-RII polyadenine sequence repeat. Among microsatellite stable (MSS) colon cancers, we now find that non-BAT-RII point mutations inactivate RII in another 15% of cases, thus doubling the known number of colon cancers in which RII mutations are pathogenetic. Functional analysis confirms that these mutations inactivate RII signaling. Moreover, another 55% of MSS colon cancers demonstrate a transforming growth factor beta signaling blockade distal to RII. The transforming growth factor beta pathway and RII in particular are major targets for inactivation in MSS colon cancers as well as in colon cancers with microsatellite instability.

Previously, we reported that unaggressive, growth factor-dependent FET human colon carcinoma cells downregulated their transforming growth factor alpha (TGFalpha) expression in a quiescent state (G0/G1) induced by growth factor and nutrient deprivation (Mulder, 1991, Cancer Res., 51:2256-2262). In contrast, highly aggressive, growth factor-independent HCT116 human colon carcinoma cells aberrantly upregulated this autocrine activity in the quiescent state (Mulder, 1991, Cancer Res., 51:2256-2262; Howell et al., 1998, Mol. Cell. Biol., 18:303-313). In this report, the role of autocrine TGFalpha and the mechanism of its regulation of expression during reentry into the cell cycle from a noncycling growth state were determined in FET cells. Optimal induction of DNA synthesis from a quiescent state in FET cells is dependent upon autocrine TGFalpha as well as exogenous transferrin and insulin. Reentry into the cell cycle resulting from treatment with exogenous transferrin and insulin resulted in approximately 3-fold induction of TGFalpha expression within 1 hr. TGFalpha induction was controlled at the transcription level, and the cis-controlling element was localized to the region between bp -370 - -201 relative to the translation start codon within the TGFalpha promoter. Thus neutralization of autocrine TGFalpha protein revealed that the induced TGFalpha autocrine activity was necessary for DNA synthesis and acted only in the early G1 phase of the cell cycle. Blockade of autocrine TGFalpha expression early in the cell cycle resulted in the reduction of DNA synthesis, whereas treatment with neutralization antibody at later times had no effect. This suggested that autocrine TGFalpha functions to initiate cell growth from noncycling states. This was further confirmed by the dependence of FET cells upon autocrine TGFalpha for colony formation in experiments where the plating density was sufficiently low to generate a lag phase in tissue culture. In contrast, TGFalpha autocrine activity was not required for exponential phase cells, as evidenced by the failure of TGFalpha neutralizing antibody to inhibit proliferation in this growth state. Taken together, these results suggest that autocrine TGFalpha acts primarily in the process of growth initiation by moving cells from a noncycling state back into the cell cycle, rather than supporting cell growth already initiated.

To analyze transforming growth factor-beta (TGF-beta) response during MCF-7 cell progression, early passage (MCF-7E, < 200 passage) and late passage (MCF-7L, > 500 passage) cells were compared. MCF-7E cells showed an IC50 of approximately 10 ng/ml of TGF-beta1, whereas MCF-7L cells were insensitive. MCF-7E cells contained approximately threefold higher levels of TGF-beta receptor type II (TbetaRII) mRNA than MCF-7L, but their TbetaRI levels were similar. MCF-7E parental cells showed higher TbetaRII promoter activity than MCF-7L cells, which could be attributed to changes in Sp1 nuclear protein levels. Receptor cross-linking studies indicated that the cell surface receptor levels parallel mRNA levels in both cell lines. Limiting dilution clones of MCF-7E cells were established to determine the heterogeneity of TbetaRII expression in this cell line, and they showed varying degrees of TbetaRII expression. Fibronectin was induced at higher levels in cells expressing higher TbetaRII levels. All three TGF-beta isoforms were detected in limiting dilution clones and parental cells, but TGF-beta1 was more abundant relative to TGF-beta2 or 3, and no correlation between TGF-beta isoform profile with TGF-beta sensitivity was found. MCF-7L cells were tumorigenic and formed xenografts rapidly and progressively, whereas MCF-7E parental and limiting dilution clonal cells showed transient tumor formation followed by regression. These results indicate that decreased TbetaRII transcription in breast cancer cells leads to a loss of TbetaRII expression, resulting in cellular resistance to TGF-beta which contributes to escape from negative growth regulation and tumor progression.

Autocrine transforming growth factor alpha (TGFalpha) activity and control mechanisms for its expression were examined in a representative clonal isolate (CBS4) of a well-differentiated human colon carcinoma cell line designated CBS. CBS4 cells expressed TGFalpha and its receptor, epidermal growth factor receptor (EGFr). Blockade of EGFr and TGFalpha by neutralizing antibodies inhibited clonal growth and the initiation of DNA synthesis from quiescence in CBS4 cells. Therefore, TGFalpha is an autocrine growth factor for CBS4 cells. Several studies have indicated that activation of the EGFr by exogenous EGF stimulates TGFalpha expression. However, in CBS4 cells EGF did not induce TGFalpha mRNA expression, indicating that EGF does not affect TGFalpha transcription in these cells. Exogenous treatment of exponentially growing cells with either EGF or EGFr blocking antibody enhanced release of TGFalpha protein into the conditioned medium. This indicated that the release of TGFalpha into the conditioned medium by exogenous EGF was at least partially due to the displacement of TGFalpha from the TGFalpha/EGFr complexes. Similarly to exponentially growing cells, the EGFr blocking antibody and EGF also enhanced TGFalpha release into the medium of CBS4 cells after release from quiescence. These results indicated that exogenous EGF had little if any effect on TGFalpha expression in these cells and suggested that TGFalpha expression might be under endogenous TGFalpha control. Blockade of the autocrine TGFalpha loop by TGFalpha neutralizing antibody suppressed TGFalpha mRNA both in exponentially growing and quiescent cells, demonstrating that autocrine TGFalpha is autoregulatory in this system.

Aberrant transcriptional regulation of transforming growth factor alpha (TGF alpha) appears to be an important contributor to the malignant phenotype and the growth factor independence with which malignancy is frequently associated. However, little is known about the molecular mechanisms responsible for dysregulation of TGF alpha expression in the malignant phenotype. In this paper, we report on TGF alpha promoter regulation in the highly malignant growth factor-independent cell line HCT116. The HCT116 cell line expresses TGF alpha and the epidermal growth factor receptor (EGFR) but is not growth inhibited by antibodies to EGFR or TGF alpha. However, constitutive expression of TGF alpha antisense RNA in the HCT116 cell line resulted in the isolation of clones with markedly reduced TGF alpha mRNA and which were dependent on exogenous growth factors for proliferation. We hypothesized that if TGF alpha autocrine activation is the major stimulator of TGF alpha expression in this cell line, TGF alpha promoter activity should be reduced in the antisense TGF alpha clones in the absence of exogenous growth factor. This was the case. Moreover, transcriptional activation of the TGF alpha promoter was restored in an antisense-TGF alpha-mRNA-expressing clone which had reverted to a growth factor-independent phenotype. Using this model system, we were able to identify a 25-bp element within the TGF alpha promoter which conferred TGF alpha autoregulation to the TGF alpha promoter in the HCT116 cell line. In the TGF alpha-antisense-RNA-expressing clones, this element was activated by exogenous EGF. This 25-bp sequence contained no consensus sequences of known transcription factors so that the TGF alpha or EGF regulatory element within this 25-bp sequence represents a unique element. Further characterization of this 25-bp DNA sequence by deletion analysis revealed that regulation of TGF alpha promoter activity by this sequence is complex, as both repressors and activators bind in this region, but the overall expression of the activators is pivotal in determining the level of response to EGF or TGF alpha stimulation. The specific nuclear proteins binding to this region are also regulated in an autocrine-TGF alpha-dependent fashion and by exogenous EGF in EGF-deprived TGF alpha antisense clone 33. This regulation is identical to that seen in the growth factor-dependent cell line FET, which requires exogenous EGF for optimal growth. Moreover, the time response of the stimulation of trans-acting factor binding by EGF suggests that the effect is directly due to growth factor and not mediated by changes in growth state. We conclude that this element appears to represent the major positive regulator of TGF alpha expression in the growth factor-independent HCT116 cell line and may represent the major site of transcriptional dysregulation of TGF alpha promoter activity in the growth factor-independent phenotype.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell growth and tumor progression. Previous work has shown that loss of functional TGF-beta type II receptor (RII) due to a frameshift mutation in the 5' half of the RII gene leads to TGF-beta resistance in a highly progressed, RER+ human colon carcinoma cell line designated HCT116. Expression of this mutated RII gene was highly repressed in RER+ cell lines such as HCT116 and RKO, as analyzed by RNase protection assays. Nuclear run-on and RII promoter-reporter (CAT) assays showed that the transcriptional levels of the RII gene in these RER+ cells were not reduced, compared to RII-expressing cells. However, the half-lives of the RII mRNA, as analyzed by RNase protection assays following actinomycin D treatment, were significantly decreased. This suggested that the decreased expression of the RII gene mutant was due to decreased mRNA stability. Furthermore, RII mRNA from HCT116 transfected with wild-type RII had a longer half-life than the endogenous mutated RII mRNA. A dominant negative RII mutant, which encodes a similarly truncated RII protein as HCT116 but lacks the extensive 3' untranslated region of RII mRNA, gave the same half-life as endogenous wild-type RII mRNA. We conclude that the frameshift mutation which results in a premature stop codon in the 5' half of the mRNA transcript accounts for the reduced RII mRNA levels in RER+ cells.

Examination of a panel of ER positive breast cancer cell lines showed that they were differentially growth inhibited by vitamin D3 and its analogue EB1089. EB1089 treatment of the breast cancer cell lines MCF-7 E, BT20, T47D, and ZR75 demonstrated a correlation between a reduction in Cdk2 kinase activity towards phosphorylation of histone H1 and a decrease in DNA synthesis, while no modulation of Cdk2 activity was observed in the vitamin D3 and EB1089 resistant cell line MCF-7 L. This was accompanied by a time dependent decrease in the percentage of S phase cells in the responsive lines. Characterization of the expression levels of Cdk2 and its related cell cycle proteins in MCF-7 E cells showed that after EB1089 treatment, there was a concentration and time dependent up-regulation of p21 as well as a decrease in cyclin A proteins. Paradoxically, cyclin E levels were increased as a function of treatment. Analysis of cyclin-Cdk2-Cdki complex formation showed that in EB1089 treated MCF-7 E cells, Cdk2, cyclin A and cyclin E immunoprecipitates contained an increased abundance of p21. In contrast to MCF-7 E cells, increases in both p21 and p27 as well as their complex formation with Cdk2 were observed in BT20 and ZR75 cells. These findings indicate that up-regulation of p21 as well as p27 in some cell types may account for the inactivation of Cdk2 activity and a G1 block of the cell cycle following EB1089 treatment.

Matrigel invasion assays were used to characterize the invasive abilities of five breast cancer cell lines. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to detect the differential gene expression of estrogen receptor (ER), E-cadherin, vimentin and cathepsin D in these cell lines. Using mRNA differential display, we identified novel cDNA clones representing the partial sequences of genes overexpressed in the invasive MDA-MB-435 cells as compared to that of the less invasive MCF-7 cells. One of the cDNAs was homologous to reticulocalbin. The studies were repeated in all of the cell lines and the overexpression of this cDNA was confirmed by RT-PRC and Northern hybridization analysis. Reticulocalbin was expressed in the highly invasive breast cancer cell lines but was not expressed in poorly invasive ones. Although its function is still unknown, reticulocalbin is implicated in tumor cell invasiveness because of its differential expression in breast tumor cell lines.

We characterized the expression of alpha 5 beta 1 integrin in two distinct phenotypes of colon carcinoma cell lines. Highly invasive colon cell lines (designated Group I cell lines) expressed higher levels of integrin alpha 5 beta 1 mRNA and protein than did poorly invasive colon cell lines (designated Group III cell lines). The relatively high expression of integrin alpha 5 beta 1 in Group I cell lines resulted in strong enhancement of cell adhesion to fibronectin (FN) tissue culture plates, whereas Group III cell lines showed little or no enhancement of cell adhesion by coating. There was no significant difference between Group I and Group III cell lines with respect to cell adhesion to laminin and collagen IV. Cell adhesion to FN in Group I cells was mainly mediated by integrin alpha 5 beta 1 because a monoclonal anti-alpha 5 subunit antibody could block cell adhesion to FN, whereas anti-alpha 2 and anti-alpha 3 antibodies had no effect on cell adhesion to FN. The divergence of alpha 5 beta 1 expression in these two distinct colon carcinoma phenotypes suggested that high expression of alpha 5 beta 1 might contribute to malignant progression in this model system. To test this hypothesis, GEO cells, a Group III cell line that did not express alpha 5 integrin, were transfected with the alpha 5 subunit. Stable transfection of alpha 5 sense cDNA into a typical GEO-limiting dilution clone led to the expression of alpha 5 subunit mRNA and cell surface alpha 5 beta 1 protein. The alpha 5 sense transfectants showed enhanced attachment to FN-coated plates and were more tumorigenic when the cells were injected into athymic nude mice. These results indicate that inappropriately high alpha 5 beta 1 integrin expression contributes to malignant progression in colon carcinoma.

Transforming growth factor beta (TGF-beta) receptor expression and signal transduction in human breast cancer are reviewed as a function of estrogen receptor (ER) expression. ER+ breast cancer cells are generally resistant to the inhibitory effects of TGF-beta. The only known exception appears to be MCF-7 early passage cells which are initially sensitive to TGF-beta, but gain resistance after long-term passage in tissue culture. A number of studies have shown that loss of sensitivity is due to inadequate TGF-beta type II (TGFRII) receptor expression. Stable transfection of TGFRII into ER+ breast cancer cell lines results in the acquisition of TGF-beta sensitivity and reversion of malignancy. Although there are exceptions, ER- breast cancer cells usually express TGFRII, but nevertheless show a low level of sensitivity to TGF-beta. Thus resistance in these cells implies a postreceptor mechanism. Given the frequency with which loss of TGF-beta sensitivity has been associated with loss of TGFRII, the ER- breast cancer cell lines may represent valuable models for identifying postreceptor mechanisms of resistance.