Semi-allogeneic dendritic cells injected via the intratumoural injection route show efficient antitumour effects in cooperation with host-derived professional antigen-presenting cells.

Dendritic cells (DC)-based immunotherapy is a potent anticancer modality. In DC-based immunotherapy, allogeneic DC may be an alternative source, but the usefulness of allogeneic DC in DC-based immunotherapy is still controversial. When used for immunotherapy, three factors may affect the efficiency of an allogeneic DC-driven antitumour response: (1) survival time, which is affected by T-cell alloresponses; (2) major histocompatibility complex incompatibility with the host cells in the context of antigen presentation; and (3) the role of host-derived professional antigen-presenting cells (pAPC). In addition, it is unclear which injection route is preferable when using allogeneic DC. In this study, we demonstrate that semi-allogeneic DC, which share half of the genes of the recipient, are more effective when used via the intratumoural (i.t.) injection route, rather than the subcutaneous (s.c.) injection route, for the induction of efficient antitumour effects and the generation of a significant tumour-specific CD8(+) T-cell response. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ. Allogeneic bone marrow transplantation (BMT) models, which permit us to separately assess the three factors described previously, show that while all three factors are important for efficient antitumour effects, the control of the alloresponse to injected DC is the most crucial for host-derived pAPC to function well when DC are administered intratumourally. This information may be useful for DC-based cancer immunotherapy under circumstances that do not allow for the use of autologous DC.

Gene expression profiles by microarray analysis during matrigel-induced tube formation in a human extravillous trophoblast cell line: comparison with endothelial cells.

Endovascular differentiation of extravillous cytotrophoblasts (EVT) during placentogenesis induces remodeling of spiral arteries that increases blood flow toward the intravillous space and is required for maintaining pregnancy. To address the molecular mechanisms involved in this differentiation, we investigated the gene expression profile during matrigel-induced tube formation in TCL1 cells, a human immortalized EVT cell line, and HUV-EC-C, human umbilical vessel endothelial cells, and compared their profiles. The numbers of genes that showed significant up-regulation (>3-fold expression at both 3 and 6h, and/or >5-fold expression at either 3 or 6h) during tube formation and significant down-regulation (0.33-fold expression at both 3 and 6h, and/or less than 0.2-fold expression at either 3 or 6h), were 969 and 659 in TCL1, respectively. In HUV-EC-C, the numbers of genes that showed significant up-regulation and down-regulation were 86 and 65, respectively. Only 73 of 1628 genes that showed significant expression changes in TCL1 were common with HUV-EC-C. The genes showing significant expression change specifically in TCL1 were associated with cellular, metabolisms, proliferation, anti-apoptosis, proteolysis adhesion, and some known to be involved in EVT differentiation or related to angiogenesis. The gene expression profile in EVT during tube formation is very different from that of endothelial cells. Further investigations based on the current data may help to elucidate mechanisms of normal and abnormal placentogenesis.

Hypoxia inducible factor 1 alpha regulates matrigel-induced endovascular differentiation under normoxia in a human extravillous trophoblast cell line.

Extravillous trophoblast (EVT) cells mimic endothelial cells during angiogenesis, inducing remodeling of the spiral arteries that increases blood flow toward the intravillous space. We have previously shown that signals involving the vascular endothelial growth factor (VEGF) axis are essential for endovascular differentiation through integrin signaling from the extracellular matrix: This was accomplished with use of the human EVT cell line TCL1, which shows tube formation that specifically recalls morphological changes in endothelial cells. To investigate endovascular differentiation in EVT further, we investigated the role of hypoxia inducible factor (HIF)1A, a subunit of HIF1 transcription factor that regulates not only adaptive responses to hypoxia, but also many cellular functions under normoxia, which was up-regulated in DNA microarray analysis during matrigel-induced endovascular differentiation under normoxia. HIF1A induces VEGF and ITGAV/ITGB3 aggregation, actions known to be important for cellular survival and endovascular differentiation in EVT. Inhibition of HIF1A up-regulation using siRNA introduction or chemical inhibition suppressed hypoxia-responsive element transcriptional activity, VEGF induction, ITGAV/ITGB3 aggregation accompanied by the inhibition of tube formation in TCL1 cells. These results suggest that HIF1A has a crucial role in regulating EVT behavior including matrigel-induced endovascular differentiation under normoxia.

Restoring movement representation and alleviating phantom limb pain through short-term neurorehabilitation with a virtual reality system.

BACKGROUND AND OBJECTIVE: We developed a quantitative method to measure movement representations of a phantom upper limb using a bimanual circle-line coordination task (BCT). We investigated whether short-term neurorehabilitation with a virtual reality (VR) system would restore voluntary movement representations and alleviate phantom limb pain (PLP). METHODS: Eight PLP patients were enrolled. In the BCT, they repeatedly drew vertical lines using the intact hand and intended to draw circles using the phantom limb. Drawing circles mentally using the phantom limb led to the emergence of an oval transfiguration of the vertical lines ('bimanual-coupling' effect). We quantitatively measured the degree of this bimanual-coupling effect as movement representations of the phantom limb before and immediately after short-term VR neurorehabilitation. This was achieved using an 11-point numerical rating scale (NRS) for PLP intensity and the Short-Form McGill Pain Questionnaire (SF-MPQ). During VR neurorehabilitation, patients wore a head-mounted display that showed a mirror-reversed computer graphic image of an intact arm (the virtual phantom limb). By intending to move both limbs simultaneously and similarly, the patients perceived voluntary execution of movement in their phantom limb. RESULTS: Short-term VR neurorehabilitation promptly restored voluntary movement representations in the BCT and alleviated PLP (NRS: p = 0.015; 39.1 ± 28.4% relief, SF-MPQ: p = 0.015; 61.5 ± 48.5% relief). Restoration of phantom limb movement representations and reduced PLP intensity were linearly correlated (p < 0.05). CONCLUSIONS: VR rehabilitation may encourage patient's motivation and multimodal sensorimotor re-integration of a phantom limb and subsequently have a potent analgesic effect. SIGNIFICANCE: There was no objective evidence that restoring movement representation by neurorehabilitation with virtual reality alleviated phantom limb pain. This study revealed quantitatively that restoring movement representation with virtual reality rehabilitation using a bimanual coordination task correlated with alleviation of phantom limb pain.

Establishment of the primary imprint of the HYMAI/PLAGL1 imprint control region during oogenesis.

Imprinting within domains occurs through epigenetic alterations to imprinting centers (ICs) that result in the establishment of parental-specific differences in gene expression. One candidate IC lies within the imprinted domain on human chromosome region 6q24. This domain contains two paternally expressed genes, the zinc finger protein gene PLAGL1 (ZAC/LOT1) and an untranslated mRNAcalled HYMAI. The putative IC overlaps exon 1 of HYMAI and is differentially methylated in somatic tissues. In humans, loss of methylation within this region is seen in some patients with transient neonatal diabetes mellitus, and hypermethylation of this region is found in ovarian cancer and is associated with changes in expression of PLAGL1, suggesting that it plays a key role in regulating gene expression. Differential methylation within this region is conserved in the homologous region on mouse chromosome 10A and is present on the maternal allele. In this paper, we report that DNA methylation is established during the growth phase of oogenesis and that this coincides with the establishment of monoallelic expression from this region lending further support to the hypothesis that this region functions as an IC.

Androgenesis as a cause of hydatidiform mole.

Q-band chromosome studies were done in 3 molar conceptuses and their parents, with special attention to six pairs of chromosomes (No. 3, 13--15, 21, and 22) in which polymorphic variants occur frequently. Those six pairs of chromosomes were uniformly homomorphic in moles, whereas at least one of them was heteromorphic in both paternal and maternal cells. Closer analyses provided evidence strongly suggesting that the moles inherited two morphologically identical haploid sets from the father and none from the mother. Thus androgenesis seemed causally related to pathogenesis of complete hydatidiform moles.

Chromosome changes in soft tissue sarcomas.

An analysis of chromosome aberrations in human tumors was performed in 29 cases of soft tissue sarcoma. The tumor tissues were disaggregated with collagenase and the cells cultured for 2-3 days. Analyzable metaphases were obtained in 15 cases, 4 of which showed only normal karyotypes. The remaining 11 tumors showed various numerical and structural abnormalities in their karyotypes: 8 tumors were near-diploid and the remaining 3 were near-triploid. G- and Q-banding analyses revealed clonal abnormalities in the 11 cases with the presence of marker chromosomes; 15 different chromosomes were involved in chromosome rearrangements, chromosomes 1 and 2 being the most frequently affected. Because of the heterogeneity of the tumor group investigated (neurogenic sarcoma, 2 liposarcomas, neurofibrosarcoma, synovial cell sarcoma, fibrosarcoma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, Ewing's sarcoma, and hemangiopericytoma), it was impossible to reach any conclusion on the specificity of the cytogenetic abnormalities for a particular tumor type.

Chromosomal changes associated with progression of the Dunning R-3327 rat prostatic adenocarcinoma system.

Several distinct sublines have developed upon serial passages of the original Dunning R-3327-H rat prostatic tumor. These tumors, each in different progressional stages, have been examined cytogenetically in the present study in order to explore the role of chromosomal changes in tumorigenesis. The original parent H tumor, from which all the other tumors were derived, has a normal karyotype, suggesting that visible alterations in chromosome structure are not essential in the early stages of the tumor. Nonrandom involvement of chromosome 4 in abnormalities observed in the increasingly aberrant tumors derived from the H tumor indicates that chromosomes most often affected may carry genetic material which is important in the regulation of cell proliferation and that this genetic material needs to undergo changes in the process of malignant transformation. Cytogenetic analysis of the Dunning sublines as a group indicated that the karyotypes of the tumors in later progressional stages tend to deviate more from the normal than those found in earlier stages. Comparison of chromosomal changes and phenotypic characteristics observed in this series of tumors suggested that the growth rate, dedifferentiation, and attainment of metastatic ability were related to the chromosome variation. The appearance and clonal development of tumor cells with a typical translocation, i.e. t(4;7), accelerated tumor growth rates of the differentiated tumors. Duplication of chromosomes was associated with a markedly increased growth rate and dedifferentiation of tumor cells in the anaplastic tumors. In addition, chromosomal loss from tetraploidy and development of complex rearrangements were associated with attainment of metastatic ability. These results point to the importance of chromosome changes in the development and biology of tumors.

Chromosome and cellular origin of choriocarcinoma.

The preferential association of choriocarcinoma with complete moles has long been recognized. In fact, of all forms of pregnancy that lead to choriocarcinoma, the risk associated with hydatidiform moles is 2000 to 4000 times greater than that of normal pregnancy or abortion. In order to corroborate the cell lineage between hydatidiform moles and choriocarcinoma, we investigated the chromosomal constitution and polymorphisms in the latter cells. Three choriocarcinomas from three women with a previous history of hydatidiform moles were examined chromosomally. Two hundred thirteen cells were analyzed for their chromosome number by conventional Giemsa staining; karyotyping of 33 cells with Q- and C-banding revealed 13 kinds of structural abnormalities unequivocally identified as of clonal origin. Two cases exhibited involvement of the long arm of chromosome 1 in rearrangements. Complete homozygosity and exclusive inheritance of a paternal genome would be expected if the tumor arose from a preceding molar pregnancy. However, the heterozygosity observed in choriocarcinoma cells suggests no common cellular lineage with complete moles; the latter originate through fertilization of an "empty egg" by a haploid sperm, followed by duplication of the chromosomes. Several possibilities have to be considered for the origin of choriocarcinomas, since it seems unlikely that alterations in the paracentric areas of the marker chromosomes occurred during tumor development. One possibility is that moles which are characterized by heterozygosity may transform into choriocarcinomas. Another possibility is that such tumors may originate from trophoblasts of the conceptuses rather than from moles.

Genetic instability coupled to clonal selection as a mechanism for tumor progression in the Dunning R-3327 rat prostatic adenocarcinoma system.

The androgen-sensitive Dunning R-3327-H prostatic adenocarcinoma has been maintained by continuous serial passage in intact male rats for many years. While it has been possible to maintain the original characteristics of the well-differentiated H tumor over 16 years, there have evolved spontaneously, however, more aberrant sublines from this tumor at several subpassages in intact male rats. Serial passage of these individual sublines has established five additional R-3327 tumors each with distinct phenotypes and each more aberrant than the parent H tumor. In addition, it has been possible by passage of the H tumor in castrated male rats to obtain a well-differentiated slow-growing adrogen-insensitive tumor termed the Hl-S tumor. The continuous serial passage of this Hl-S tumor has likewise resulted in the emergence of three new types of Dunning tumors. The results from the biochemical and chromosomal studies presented demonstrate that there is a consistent association in each of these tumor progressions between the expression of genetic instability, which results in the addition of phenotypically new clones of cells to the tumor population, and the subsequent selection of these newly developed clone. These results suggest that the process of genetic instability coupled to clonal selection is one mechanism for the change in tumor phenotype characteristically associated with tumor progression within this system of prostatic tumors.

Evidence for multiple pathways to cellular senescence.

Normal cells in culture generally senesce whereas tumor-derived cells are often, but not without exception, immortal and grow indefinitely. For cells to escape the senescence program, normal genes must be lost or inactivated as shown by somatic cell genetic studies. For example, the introduction of specific chromosomes by microcell-mediated chromosome transfer has been shown to induce senescence of human and rodent tumor cell lines, and the mapping of over ten senescence genes has been achieved by this method. In this study, we observed that two different normal chromosomes induce senescence in the same human endometrial carcinoma cell line, which suggests that multiple pathways to senescence are inactivated in this cell line. This hypothesis has implications for the mechanisms of cellular senescence and its role in carcinogenesis. Furthermore, this hypothesis can explain why not all tumor-derived cells are immortal.

Specific cytogenetic changes in ovarian cancer involving chromosomes 6 and 14.

Cytogenetic studies were performed in 12 papillary serous adenocarcinomas of the ovary. Of the more than 19 clonal structural chromosome abnormalities observed in these cancers, 6q- and 14q+ were found to be the most frequent. Both markers coexisted in the cells of eight cases; in the other four cases, either a 6q- or 14q+ was present. In at least six cases, the additional segment on the long arm of chromosome 14 appeared to originate, on the basis of the chromosomal quantity and fluorescence pattern, from the missing part of chromosome 6. This suggested that the 6q- and 14q+ markers had arisen as a result of a reciprocal translocation at Bands q21 and q24, respectively, i.e., t(6;14)(q21;q24). However, it is uncertain in the remaining six cases whether an identical type of translocation was responsible for the formation of the markers. Thus, abnormalities involving chromosomes 6 and 14 seem to be specifically associated with papillary serous adenocarcinoma of the ovary.

Malignant potential of homozygous and heterozygous complete moles.

There are two main mechanisms of origin for complete hydatidiform mole: (a) fertilization of an empty egg by a haploid sperm followed by duplication (monospermic mole); and (b) fertilization of such an egg by two haploid spermatozoa (dispermic mole). The former is inevitably homozygous (homozygous mole), whereas the latter may be heterozygous for a given genetic marker (heterozygous mole). A recent cytogenetic study showed that three cases of choriocarcinoma were undoubtedly heterozygous, which prompted us to compare the incidence of postmolar sequelae between patients with homozygous moles and those with heterozygous moles. Making use of chromosomal heteromorphisms and human lymphocyte antigen and phosphoglucuromutase 1 polymorphisms, we established the androgenetic origin of a complete mole in 49 of 56 cases. Homozygosity was confirmed in 21 moles, and heterozygosity was confirmed in five. Three of five patients with heterozygous moles developed postmolar trophoblastic disease, whereas none of the 21 patients with homozygous moles suffered postmolar trophoblastic disease except one who showed signs of degenerating residual trophoblasts. Consistent with this observation is the fact that all of the four destructive moles studied here were of dispermic origin. Thus, heterozygous moles seem to have a higher malignant potential than do homozygous moles.

Analysis of specific gene mutations in the transforming growth factor-beta signal transduction pathway in human ovarian cancer.

Several proteins, including transforming growth factor beta (TGF-beta) receptor type I (RI), TGF-beta receptor type II (RII), Smad2, Smad3, and Smad4/DPC4, have been identified in the transduction pathway of the tumor suppressor TGF-beta. Mutations in TGF-beta RI, TGF-beta RII, Smad2, and Smad4/DPC4 genes are associated with several human cancers. The present study examines these gene mutations in 32 human ovarian cancers and 14 patient-matched normal tissues. For the first time, mutations in the Smad2 and Smad4 genes were analyzed in relation to human ovarian cancer. Gene mutations of TGF-beta RI, TGF-beta RII, Smad2, and Smad4 were analyzed using specific primers by PCR-single-strand conformational polymorphism (SSCP), and the results revealed a frameshift mutation at codons 276-277 (CTCTGG-->CTGCGTGG) in exon 5 of TGF-beta RI in 10 of 32 tumor samples (31.3%). This mutation was associated with reduced or absent expression of TGF-beta RI protein and p53 protein in tumor tissues. We detected SSCP variants of TGF-beta RII in exon 2 in 20 of 32 tumors. Sequence analysis of these variants revealed an A to G transition at the seventh band of intron 2. In this A to G polymorphism in intron 2, 12 samples (37.5%) had A/A alleles, 12 (37.5%) had A/G alleles, and 8 (25%) had G/G alleles. We detected Smad2 SSCP variants in exon 4 in 12 of 32 tumors (37.5%). Sequence analysis revealed a 2-bp deletion in the polypyrimidine tract of intron 3, which is located at position -39 to -56 in the splice acceptor site of the intron 3-exon 4 junction. No SSCP variants were detected in the Smad4 gene. These findings suggest that mutations in the TGF-beta RI and in its signal transduction pathway are likely responsible for human ovarian carcinogenesis.

Multiple chromosomes carrying tumor suppressor activity for a uterine endometrial carcinoma cell line identified by microcell-mediated chromosome transfer.

Putative tumor suppressor genes can be mapped to specific chromosomes by the introduction of individual chromosomes derived from normal cells via microcell fusion. We have examined whether a highly malignant human uterine endometrial carcinoma cell line, HHUA, can be suppressed by only one normal chromosome or by multiple chromosomes. A library of mouse A9 clones containing different human chromosomes tagged with the pSV2-neo plasmid DNA were constructed. Transfer by microcell fusion of either chromosome 1, 6, 9, 11, or 19 into the HHUA tumor cell line was performed, and the abilities of the microcell hybrids to form tumors in nude mice were examined. The introduction of a chromosome 19 had no effect on the tumorigenicity of the cells, whereas microcell-hybrid clones with an introduced chromosome 1, 6 or 9 were completely suppressed for tumorigenicity. A decrease in tumor-take incidence in some but not all clones was observed following the introduction of a chromosome 11. The nontumorigenic microcell hybrids with an introduced chromosome 1 differed from the nontumorigenic microcell hybrids with an introduced chromosome 6, 9, or 11. A large percentage of hybrids with chromosome 1 senesced and/or showed alterations in cellular morphology and transformed growth properties in vitro. No growth or morphology alterations were observed following transfer of the other chromosomes. These results may indicate that more than one chromosome carries a tumor suppressor gene(s) for this human uterine endometrial carcinoma cell line and support the hypothesis that multiple tumor suppressor genes control the tumorigenic phenotype in the multistep process of neoplastic development.

Contribution of enhanced transcriptional activation by ER to [12Val] K-Ras mediated NIH3T3 cell transformation.

We investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation by the [12Val] K-Ras mutant. This mutant enhanced the steady state level of ER. Cells expressing mutant K-Ras (K12V cell) were tumorigenic. To determine the role of ER accumulation in Ras-transformed cells, we developed cells (KwtER cells) that overexpressed both wild-type (wt) K-Ras and ER, and found these cells were also tumorigenic. E2 stimulated the transcriptional activity by ER dominantly in K12V cells. However, only partial activation of ER by E2 was seen in KwtER cells. In the presence of 10% serum in media, the activation of ER appeared only in transformed KtwER and K12V cells, suggesting that two independently transmitted signals, the E2-ER binding and the ER-AF1 activation, are necessary for ER activation and that the dominant activation of ER might be involved in Ras-mediated cell transformation. Co-expression of progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of the activation of ER. The antisense oligomers complementary to the ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.

Human chromosome 7 carries a putative tumor suppressor gene(s) involved in choriocarcinoma.

Choriocarcinoma developed from a complete hydatidiform mole has an unique genetic feature that involves monoallelic contribution from the paternal genome. To determine the chromosome carrying putative tumor suppressor gene(s), microcell-hybrids were isolated following fusion of choriocarcinoma cells with microcells from mouse A9 cells containing a single human chromosome (1, 2, 6, 7, 9 or 11). Microcell-hybrids with the introduction of chromosome 7 were suppressed or modulated for tumorigenicity and exhibited altered in vitro growth properties. Introduction of chromosomes 1, 2, 6, 9 or 11 had no effect. Tumorigenic revertants isolated from microcell-hybrids with the introduced chromosome 7 contains reduced numbers of chromosome 7. These findings suggest that chromosome 7 contains a putative tumor suppressor gene(s) for choriocarcinoma. Alterations in tumorigenic phenotypes seen in microcell-hybrids were not associated with the presence of either ERV3 or H-plk locus located on the introduced chromosome 7, indicating the putative tumor suppressor gene(s) is outside of ERV3 and H-plk gene loci. Furthermore, we obtained evidence to define a critical region on chromosome 7 (7p12-7q11.23) that was frequently lost in surgically removed choriocarcinoma tissues and cell lines. Using a panel of microsatellite markers, biallelic deletions were observed, which strongly suggests the presence of a tumor suppressor gene(s) within this critical region.

Long-term results of the Kumamoto Study on optimal diabetes control in type 2 diabetic patients.

OBJECTIVE: To examine whether intensive glycemic control could decrease the frequency or severity of diabetic microvascular complications, an 8-year prospective study of Japanese patients with type 2 diabetes was performed. RESEARCH DESIGN AND METHODS: A total of 110 patients with type 2 diabetes (55 with no retinopathy [the primary prevention cohort] and 55 with simple retinopathy [the secondary intervention cohort]) were randomly assigned to multiple insulin injection therapy (MIT) groups and administered three or more daily insulin injections or assigned to conventional insulin injection therapy (CIT) groups and administered one or two daily intermediate-acting insulin injections. Worsening of microvascular complications was regularly assessed during 8 years. Two or more steps up in the 19 stages of the modified Early Treatment of Diabetic Retinopathy Study classification in retinopathy and one or more stages up among three stages in nephropathy (normoalbuminuria, microalbuminuria, and albuminuria) were defined as worsening of complications. RESULTS: In both primary prevention and secondary intervention cohorts, the cumulative percentages of worsening in retinopathy and nephropathy were significantly lower (P < 0.05) in the MIT group than in the CIT group. In neurological tests after 8 years, the MIT group showed significant improvement (P < 0.05) in the median nerve conduction velocities (motor and sensory nerves), whereas the CIT group showed significant deterioration (P < 0.05) in the nerve conduction velocities and vibration threshold. From this study, the glycemic threshold to prevent the onset and progression of diabetic microvascular complications was as follows: HbA1c < 6.5%, fasting blood glucose concentration < 110 mg/dl, and 2-h postprandial blood glucose concentration < 180 mg/dl. CONCLUSIONS: Intensive glycemic control can delay the onset and progression of the early stages of diabetic microvascular complications in Japanese patients with type 2 diabetes.

Oncogenic Ras modulates epidermal growth factor responsiveness in endometrial carcinomas.

Since the majority of endometrial carcinomas do not contain any detectable ras mutations, the precise contribution of aberrant Ras function, if any, to endometrial carcinoma development remains to be determined. Since there is considerable evidence that Ras transformation is associated with a decreased requirement for growth factors, we compared the growth response of endometrial carcinoma cells harbouring wild-type (Ishikawa cells) or mutated (HHUA cells) K-ras to epidermal growth factor (EGF). K-ras mutation did not significantly affect the level of the EGF receptor (EGFR) expressed in these carcinoma cells. EGF could stimulate the growth of Ishikawa, but not HHUA cells. Furthermore, EGF caused elevation of Ras-GTP levels in Ishikawa, but not HHUA cells. However, the introduction of mutated, but not normal, K-ras into Ishikawa cells rendered them non-responsive to EGF growth stimulation. Thus, the presence of mutated K-ras alone modulated the growth response of endometrial carcinoma cells to EGF. An inhibitor of the EGFR tyrosine kinase activity could prevent soft agar colony formation of Ishikawa cells, but not HHUA or mutant K-ras(12V)-transfected Ishikawa cells. Taken together, these results suggest that mutated K-ras causes a loss of responsiveness to EGF stimulation and that EGFR function is dispensable for the growth of mutant Ras-positive endometrial carcinoma cells.