Phase I study of E7010

E7010 is a novel sulfonamide which was discovered using slow-growing colon 38 carcinoma cells as a screening model. E7010 exhibits a broad spectrum of antitumor activity against human tumor xenografts. The mechanism of action is by arresting the progression of cells in M phase of the cell cycle by inhibiting tubulin polymerization. The objective of this phase I study was to determine the maximum allowable dose (MAD), toxicity, and pharmacokinetics of single or 5-day repeated doses of E7010. In the single-dose study, E7010 was administered orally to 16 patients at doses ranging from 80 to 480 mg/m². The dose-limiting toxicity was peripheral neuropathy at a dose of 480 mg/m². Hematological and gastrointestinal toxicities were mild. In the 5-day repeated-dose study, 41 patients were given E7010 at doses ranging from 30 to 240 mg/m² per day. The dose-limiting toxicities were peripheral neuropathy and intestinal paralysis. Gastrointestinal toxicity was dose-dependent but not severe. Hematological toxicity was not dose-dependent. Pharmacokinetic analysis in the single-dose study showed a rapid increase in the plasma levels of the drug after administration, followed by disappearance with a t_1/2 of 4.4–16.6 h. The variation in area under the plasma concentration-time curve (AUC) between the patients was small and increased in a dose-dependent manner. Total drug recovery in urine 72 h after administration was 77.8 ± 11.4%, indicating that E7010 has favorable absorption and elimination profiles. The changes in the plasma levels of E7010 on day 5 in the 5-day repeated-dose study were almost the same as those on day 1, indicating that the drug did not accumulate. In the single-dose study, spinal cord metastasis exhibited a 74% reduction in a patient with uterine sarcoma and a minor response (MR) was observed in a pulmonary adenocarcinoma patient. In the 5-day repeated-dose study decreases in the tumor markers carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC) were observed in a patient with stomach cancer and in a patient with recurrent uterine cervical carcinoma, respectively. The recommended phase II doses are 320 mg/m² for a single-dose study and 200 mg/m² per day for a 5-day repeated-dose study. Since the activity of E7010 is time-dependent, i.e. a certain concentration of E7010 is required for more than 12 h to suppress the growth of P388 leukemia cells, it is recommended that subsequent phase I/II studies be conducted using a divided dose schedule in order to maintain the blood level of E7010. Received: 12 February 1997 / Accepted: 6 November 1997     

Brefeldin A Blocks the Cytotoxicity of T Cell Receptor α/β and γ/δ Cytotoxic T Lymphocyte Clones Reacting against Human Autologous Cancer Cells

We studied the effector mechanism of T cell receptor (TCR) α/β - and γ/δ -type cytotoxic T lymphocyte (CTL) clones that react with human autologous tumor cells. Treatment of tumor cells with a fungal antibacterial reagent, brefeldin A (BFA), resulted in the inhibition of cytotoxicity of an autologous tumor (HST-2)-specific CD8⁺ TCR α/β -type CTL, TcHST-2. Other anti-metabolites such as chloroquine, cycloheximide and colchicine did not affect the cytotoxicity. The cell-surface antigen expression, including MHC class I molecules, was not influenced by BFA treatment. Furthermore, BFA did not influence the cytotoxicity of lymphokine-activated killer cells and natural killer cells. Since BFA blocks the transport of peptides from endoplasmic reticulum to the Golgi apparatus, the above data suggest that BFA could affect washing out of the peptide fragments from the MHC class I groove. Consequently, target tumor cells were protected from killing by CTL, Moreover, we obtained a CD4⁻, 8⁻, TCR γ/δ -type (Vδ1⁺) CTL clone, TcHOT, that reacts against an autologous ovarial carcinoma, HOT. BFA could also inhibit this cytotoxicity, and it is likely that different presenting molecules other than MHC class I proteins participate in the cytotoxicity of this TCR gm/δ - type CTL. These studies suggest that both TCR α/β - and γ/δ -type CTL may require antigenic peptides that are most likely derived from the BFA-sensitive, intracellular endogenous target proteins.     

β-Catenin Accumulation and Mutation of Exon 3 of the β-Catenin Gene in Hepatocellular Carcinoma

study was conducted to clarify the contribution of β-catenin accumulation and mutation of the β- catenin gene to hepatocarcinogenesis. β-Catenin accumulation was examined immunohistochemically in 38 paired samples of hepatocellular carcinoma (HCC) and corresponding non-cancerous liver tissue. Gene mutation was analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing using intronic primers encompassing exon 3. Neither accumulation nor mutation was detected in non-cancerous liver tissues that showed no remarkable histological features, chronic hepatitis or liver cirrhosis. Accumulation of β-catenin was seen in the nucleus, cytoplasm or cell membrane in 15 of 38 (39%) HCC samples, and gene mutation was seen in 9 of 38 (24%) HCC samples. Although there was a significant correlation between accumulation and mutation ( P <0.01), six HCCs without mutation also showed accumulation. Samples of early HCC showed neither accumulation nor mutation, and accumulation and mutation were each correlated significantly with portal vein tumor involvement ( P <0.05). The present results indicate that (1) mutation of exon 3 of the β- catenin gene can lead to β-catenin accumulation, although other mechanisms of accumulation may also operate in HCC, and (2) β- catenin accumulation and mutation of the β- catenin gene are not early events in hepatocarcinogenesis, and may be associated with the malignant progression of HCC.     

Inhibition of Tumor Necrosis Factor-α and β Secretion by Lymphokine Activated Killer Cells by Transforming Growth Factor-β

Transforming growth factor- β (TGF- β ) has a variety of immunosuppressive properties. We investigated the effect of TGF- β secreted by glioblastoma (T98G) cells on the secretion of tumor necrosis factor- α and - β (TNFs) by lymphokine activated killer (LAK) cells stimulated with tumor cells. The supernatant from T98G cells was preincubated with anti-TGF- β l and - β 2 neutralizing antibodies or untreated, and added to a coculture of LAK and Daudi cells. The neutralizing antibodies were added to LAK/Daudi and LAK culture, and natural human TGF- β and recombinant human TGF- β were also added to the LAK/Daudi culture. LAK cells were also cultured with T98G cells, of which the supernatant contained both active and latent forms of TGF-/ β 1 and TGF- β 2, and the neutralizing antibodies were added to the coculture. TNFs activity in the supernatants from LAK/ Daudi cultures was examined by a specific bioassay. Addition of the supernatant from T98G cells to LAK/Daudi culture resulted in the inhibition of TNFs secretion by LAK cells. The inhibition was abrogated by the pretreatment of the supernatants with the anti-TGF- β antibodies. Addition of TGF- β and TGF- β to LAK/Daudi culture inhibited TNFs secretion by LAK cells in a dose-dependent manner. Addition of anti-TGF β - antibodies to LAK culture resulted in an increase of TNFs secretion. These results suggest that, if tumor cells have the capacity to convert TGF- β from a latent to an active form, the active TGF- β suppresses TNFs secretion by LAK cells stimulated with the tumor cells, and that TGF- β secreted and activated by glioblastoma cells suppresses the propagation of immune reaction by inhibiting TNFs secretion by activated lymphocytes adjacent to tumor cells.     

Effect of E7010 on liver metastasis and life span of syngeneic C57BL/6 mice bearing orthotopically transplanted murine Colon 38 tumor

PURPOSE: E7010 is an orally active sulfonamide antitumor agent showing good activity against various subcutaneously inoculated rodent tumors and human tumor xenografts. The purpose of this study was to evaluate the effect of E7010 on liver metastasis and life span of mice bearing orthotopically transplanted murine Colon 38 tumor. METHODS: Orthotopic transplantation of murine Colon 38 tumor as intact tissue yielded hepatic metastasis with a high incidence in about 1 month in C57BL/6 mice, and the mice died in about 2 months with cachexia. In this model, the maximum tolerated dose of E7010 (100 mg/kg per day) was administered orally on various schedules, including for 14 days or daily until death, starting at 14 days after transplantation, or for 8 days from 21 days after transplantation. RESULTS: E7010 showed tumor growth inhibition (T/C=40%) at the orthotopic site similar to that at the subcutaneous site (T/C = 32%) when administered from 14 days after transplantation. When E7010 was started from 21 days after transplantation, it significantly decreased the number of hepatic metastases (control 17.1+/-20.8, E7010 2.6+/-5.3), although inhibition of tumor growth at the orthotopic site was only moderate (T/ C=60%). The administration of E7010 until death produced a significant increase in life span (control 49.8+/-8.9 days, E7010 62.5+/-6.1 days). Although the tumor weight of the E7010-treated group on the day of death was similar to that of the untreated group (control 1.166+/-0.507 g, E7010 1.211+/-0.632 g), there were significantly fewer liver metastases in the E7010-treated group (control 41.3+/-31.1, E7010 2.0+/-2.0). CONCLUSION: E7010 suppressed tumor growth at both primary and metastatic sites and increased life span in an orthotopic transplantation model of murine Colon 38 tumor in syngeneic C57BL/6 mice. Hepatic metastasis was inhibited more effectively than the growth of the primary tumor.     

Estrone and 17β-Estradiol Reverse Breast Cancer Resistance Protein-mediated Multidrug Resistance

Breast cancer resistance protein (BCRP), an adenosine triphosphate-binding cassette transporter, confers resistance to a series of anticancer reagents, including mitoxantrone, SN-38 and topotecan. In the present study, we found that estrone and l7β-estradiol potentiated the cytotoxicity of mitoxantrone, SN-38 and topotecan in BCRP-transduced K562 cells (K562/BCRP). These estrogens showed only a marginal effect, or none, in parental K562 cells. Estrone and 17β-estradiol increased the cellular accumulation of topotecan in K562/BCRP cells, but not in K562 cells, suggesting that these estrogens inhibit the BCRP-mediated drug efflux and overcome drug resistance.     

Differential regulation of the cytotoxicity activity of paclitaxel by orobol and platelet derived growth factor in human ovarian carcinoma cells

Paclitaxel (PX) binds to and stabilizes tubulin, preventing depolymerization, and resulting in cell death. Based on a previous report showing the activity of phosphatidylinositol kinase (PIK) on tubulin, we investigated the effect of the PI4K inhibitor orobol and the PI3K activator platelet derived growth factor (PDGF) on PX sensitivity. Drug sensitivity was examined by classical colony forming assay. Tubulin isotype expression was determined by semi-quantitative RT-PCR. Microtubule texture was observed by laser confocal microscope using anti-beta-tubulin antibody. Apoptotic activity was estimated by frequency of condensed nuclear chromatin with Hoechst 33342 stain. Orobol enhanced PX sensitivity of human ovarian carcinoma 2008 cells by 18.9+/-1.2-fold (N=3; P<0.01). In contrast, pretreatment with PDGF rendered cells resistant to PX by 2.3+/-0.4-fold (N=3; P<0.01). Neither orobol nor PDGF showed any effect on cell growth. Orobol produced a 2.5-fold sensitization in cisplatin-resistant 2008/C13*5.25 (C13) cells, and PDGF rendered the cells 2.3-fold resistant to PX. Orobol suppressed the beta 4a-tubulin isotype expression by 85% and other isotypes by 20%. In contrast, PDGF induced beta 4a-tubulin isotype expression by 1.3-fold, while it supressed all the other isotypes by 20-40%. Orobol produced thick microtubules and PDGF generated ring condensed microtubules. Orobol promoted PX-induced apoptosis, while PDGF caused 50% reduction of apoptosis. These results indicate that orobol and PDGF regulate PX sensitivity by reciprocally altering the proportion of tubulin isotype expression and PX-induced apoptotic signaling.     

Modulation of c-myc Expression by Transforming Growth Factor β1 in Human Hepatoma Cell Lines

The effects of transforming growth factor β1 (TGF-β1) on cell proliferation of human hepatoma cell lines, PLC/PRF/5 and Mahlavu, were investigated under serum-free conditions. DNA synthesis was strongly inhibited in the PLC/PRF/5 cells by addition of TGF-β1 (0.5 to 4.0 ng/ml), but remained unchanged in the Mahlavu cells. Also the expression of c- myc mRNA was suppressed by the addition of TGF-β1 in the PLC/PRF/5 cells but not in the Mahlavu cells. These results indicate that TGF-β1 might regulate cell growth, in part, by modulating c- myc expression, although there is no direct proof that c- myc expression is really relevant to DNA synthesis mediated by TGF-β1.     

High Expression of Uridine Diphosphate-galactose: Lc3Cer βl-3 Galactosyltransferase in Human Uterine Endometrial Cancer-derived Cells as Measured by Enzyme-linked Immunosorbent Assay and Thin-layer Chromatography-immunostaining

We have developed a new procedure for the selective determination of β1-3 and β1-4 galactosyltrans-ferases with Lc₃Cer as the substrate and the microsomes of fetal and adult porcine livers as the enzyme sources. This method was based on the detection of such products as Lc₄Cer for β1-3 galactosyltransferase (β1-3GT) and nLc₄Cer for β1-4 galactosyltransferase (β1-4GT), with monoclonal anti-Lc₄Cer and anti-nLc₄Cer antibodies, respectively. This method thus enabled us to differentiate the activity of β1-3GT from that of 4bT1-4GT with a high degree of sensitivity. The method was then used to determine the activities of both enzymes in human gynecological carcinoma-derived cells. Four of the five cell lines derived from uterine endometrial cancer expressed significantly high levels of specific activity of β1-3GT among the cell lines examined, while their β1-4GT activities were less than 20% of that for β1-3GT in the endometrial carcinoma-derived cells. On the other hand, a higher specific activity of β1-4GT than that of β1-3GT was detected in the cell lines derived from uterine cervical and ovarian cancers. These findings were thus found to correlate closely with the rate of expression of Lc₄Cer- and nLc₄Cer-based carbohydrate chains in the cell lines based on the results of immunohistochemical staining.     

Up-regulation of integrin β3 in radioresistant pancreatic cancer impairs adenovirus-mediated gene therapy

Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower ( P  < 0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin β3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin β3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin β3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy. ( Cancer Sci  2009; 100: 1902–1907)     

Mutations of the β-Catenin Gene in Endometrial Carcinomas

To investigate the contribution of β-catenin to the development of endometrial carcinoma, we searched for genetic alterations of the β-catenin gene in primary endometrial carcinomas. Mutational analysis of exon 3 of the β-catenin gene, encoding the serine/threonine residues for GSK-3β phosphorylation, was performed for 35 tumors. Nucleotide sequencing analysis revealed that 5 tumors (5/35, 14%) contained mutations (S33C, S37C, S37F, T41A) that altered potential GSK-3β phosphorylation sites. Each of the mutations resulted in the substitution of serine/threonine residues that have been implicated in the down-regulation of β-catenin through phosphorylation by GSK-3β kinase. Furthermore, the incidence of β-catenin mutations was significantly higher in early-onset (3 of 5) than that in late-onset tumors (2 of 30) (P=0.014, Fisher's exact test). Replication error (RER)-positive phenotype was not detected in tumors with the β-catenin gene mutation, although 10 of 35 tumors revealed RER. We performed immunohistochemistry of β-catenin in 17 cases for which tissue samples were available. We confirmed accumulation of β-catenin protein in both the nucleus and cytoplasm in 3 tumors, including two in which amino acid alterations had occurred at codon 33 and 37. The other case had no mutation in exon 3. Our results suggested that mutations at serine/threonine residues involved in phosphorylation by GSK-3β affected the stability of β-catenin. Accumulation of mutant β-catenin could contribute to the development of a subset of endometrial carcinomas, particularly those of the early-onset type.     

Antitumor Effect of Interleukin-1β in the Double Grafted Tumor System

The antimetastatic effect of recombinant human interleukin-1β (rIL-1β) in a new experimental mouse model was studied. Intratumoral administration of IL-1β strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumor. Subsequently, the anti-metastatic effect of IL-1β was examined in the double grafted tumor system, in which mice first received simultaneous intradermal inoculations of Meth-A in both right (10⁶ cells) and left (2×10⁵ cells) flanks and were then injected with 0.2 μg of IL-1β in the right tumor on days 3, 4 and 5. IL-1β significantly inhibited the growth of the left, non-treated tumor. When mice received only an inoculation of Meth-A (2×10⁵ cells) in the left flank and were injected subcutaneously with IL-1β into the right flank on day 3 (single tumor system), there was no inhibition of the growth of the left, non-treated tumor. These findings suggest that intratumoral IL-1β immunotherapy in one region has an effect on tumor growth in another region. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of IL-1β. Adoptive transfer of the immunized spleen cells caused the complete regression of Meth-A tumors. These results suggest that intratumoral administration of IL-1β might induce cytotoxic cells in the left non-treated tumor of the double grafted tumor system and bring about the regression of metastatic tumors. On the other hand, recombinant tumor necrosis factor was effective only on the treated, right tumor, having no effect on the distant, left tumor in the double grafted tumor system. Recombinant interleukin-2 was effective on neither the right tumor nor the left tumor in this system. These results show that there are major differences of antitumor mechanism among cytokines.     

Mechanism of inhibition of tumor angiogenesis by β-hydroxyisovalerylshikonin

Shikonin and β-hydroxyisovalerylshikonin (β-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that β-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. β-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using β-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. β-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. β-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and β-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling. ( Cancer Sci 2009; 100: 269–277)     

β-Hydroxyisovalerylshikonin Is a Novel and Potent Inhibitor of Protein Tyrosine Kinases

β-Hydroxyisovalerylshikonin (β-HIVS), a compound isolated from Lithospermium radix , most efficiently induced cell-death in two lines of lung cancer cells, namely, NCI-H522 and DMS114, whereas shikonin was effective against a wide variety of tumor cell lines. During our studies of the mechanism of action of β-HIVS on tumor cells, we found that this compound inhibited protein tyrosine kinase (PTK) activity. The tyrosine kinase activities of a receptor for EGF (EGFR) and v-Src were strongly inhibited and that of KDR/Flk–1 was weakly inhibited by β-HIVS. The inhibition by β-HIVS of the activities of EGFR and v-Src was much stronger than that by shikonin. The IC₅₀values of β-HIVS for EGFR and v-Src were approximately 0.7 μM and 1 μM , respectively. Moreover, the inhibition of v-Src by β-HIVS was non-competitive with respect to ATP. These results strongly suggest that the action of β-HIVS, as well as that of shikonin, involves the inhibition of PTK, and they also suggest the possibility of producing a novel group of PTK inhibitors based on shikonin as the parent compound.     

Mutational Analysis of β-Catenin Gene in Japanese Ovarian Carcinomas: Frequent Mutations in Endometrioid Carcinomas

To investigate the contribution of the β-catenin gene to the development of ovarian carcinomas, mutational analysis of exon 3 of the β-catenin gene was conducted. We analyzed 61 primary ovarian carcinomas, consisting of 49 non-endometrioid-type and 12 endometrioid-type tumors, for genetic alteration of the β-catenin gene. Five carcinomas showed β-catenin mutations (S37C, T41I, T41A), including 4 (33%) of 12 endometrioid-type tumors and 1 (14%) of 7 mucinous-type tumors. All of these mutations altered at the serine/threonine residues that are potential sites of GSK3-β phosphorylation. We detected no carcinomas with interstitial deletion involving exon 3 of β-catenin. Furthermore, we immunohistochemically studied 27 of the 61 ovarian carcinomas. Both nuclear and cytoplasmic β-catenin expressions were demonstrated in 4 of the 27 ovarian carcinomas for which tissue samples were available for examination. All 4 cases exhibited mutations in exon 3 of β-catenin , including a mucinous carcinoma. Our results suggested that β-catenin gene mutation at potential GSK3-β phosphorylation sites results in accumulation of β-catenin protein within the cells and its translocation to nuclei. Accumulated β-catenin protein may be involved in the development of endometrioid-type ovarian carcinomas, and some mucinous-type ovarian carcinomas.     

Frequent β-Catenin Abnormalities in Bone and Soft-tissue Tumors

We have screened mutations of the β-catenin gene by using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method in 62 malignant bone and soft-tissue tumors, including malignant fibrous histiocytomas (MFHs), osteosarcomas, synovial sarcomas, liposarcomas, malignant schwannomas, and other types of tumors, as well as 11 benign tumors. β-Catenin-activating missense mutations were found in two malignant tumors. One found in MFH occurred at codon 45 and caused an amino acid substitution from serine (one of the GSK3β-targeted phosphorylation sites) to phenylalanine. The other, detected in synovial sarcoma at codon 32, resulted in an amino acid change from aspartic acid (located adjacent to the phosphorylation target, serine, encoded by codon 33) to tyrosine. Furthermore, we found accumulation of β-catenin by western-blotting analysis in 12 of 19 malignant tumors in which we found no mutation involving exon 3. Our results suggested the possible involvement of β-catenin activation, by β-catenin gene mutation or alteration of other factor(s), in the formation and/or progression of various types of bone and soft-tissue tumors.     

β class II tubulin predominates in normal and tumor breast tissues

Background

Antimitotic chemotherapeutic agents target tubulin, the major protein in mitotic spindles. Tubulin isotype composition is thought to be both diagnostic of tumor progression and a determinant of the cellular response to chemotherapy. This implies that there is a difference in isotype composition between normal and tumor tissues.

Methods

To determine whether such a difference occurs in breast tissues, total tubulin was fractionated from lysates of paired normal and tumor breast tissues, and the amounts of β-tubulin classes I + IV, II, and III were measured by competitive enzyme-linked immunosorbent assay (ELISA). Only primary tumor tissues, before chemotherapy, were examined. Her2/neu protein amplification occurs in about 30% of breast tumors and is considered a marker for poor prognosis. To gain insight into whether tubulin isotype levels might be correlated with prognosis, ELISAs were used to quantify Her2/neu protein levels in these tissues.

Results

β-Tubulin isotype distributions in normal and tumor breast tissues were similar. The most abundant β-tubulin isotypes in these tissues were β-tubulin classes II and I + IV. Her2/neu levels in tumor tissues were 5–30-fold those in normal tissues, although there was no correlation between the Her2/neu biomarker and tubulin isotype levels.

Conclusion

These results suggest that tubulin isotype levels, alone or in combination with Her2/neu protein levels, might not be diagnostic of tumorigenesis in breast cancer. However, the presence of a broad distribution of these tubulin isotypes (for example, 40–75% β-tubulin class II) in breast tissue, in conjunction with other factors, might still be relevant to disease progression and cellular response to antimitotic drugs.     

Growth Inhibition, Enhancement of Intercellular Adhesion, and Increased Expression of Carcinoembryonic Antigen by Overexpression of Phosphoinositides-specific Phospholipase C β1 in LS174T Human Colon Adenocarcinoma Cell Line

By using a retrovirus-derived system we generated derivatives of the human colon adenocarcinoma cell line LS174T (ATCC CL 188) that stably overexpress a full-length cDNA encoding the β1 isoform of bovine phosphoinositides-specific phospholipase C (PI-PLC). This was confirmed by the elevated levels of catalytic activity to release phosphoinositides from phosphatidylinositol (PI-PLC) or phosphatidylinositol-bis-phosphate (PIP₂-PLC), and the enhanced expressions of messenger RNA and protein. PI-PLC β1 overexpresser clones grew to form cell clumps floating in liquid medium, whereas the pMV7-introduced control clones displayed morphologic characteristics that were very similar to those of the parent LS174T cell line. Three individual PI-PLC β1 overexpresser cell lines displayed increased doubling time (18.0 h, 21.5 h, and 23.8 h) when compared with 4 individual pMV7-introduced control cell lines (13.1 h, 10.7 h, 12.9 h, and 9.3 h). Anchorage-independent growth ability in soft agar medium was dramatically suppressed by overexpression of PLC β1, and the ability of PLC-overproducer clones to form aggregates when cultured in liquid medium was dramatically enhanced when compared with that of pMV7-introduced control clones. Tumorigenicity of PLC β1-overproducers was much weaker than that of vector-transduced control clones. The spontaneous release of carcinoembryonic antigen from PLC β1-overproducer clones was much higher than that from pMV7 control clones. The ability of PLC β1-overproducer clones to form aggregates during suspension culture was much stronger than that of the control clones. These results provide the first evidence that elevated levels of endogenous PI-PLC β1 suppress tumor cell growth, but enhance the ability to form cell aggregates and to release carcinoembryonic antigen, an intercellular adhesion molecule.