Interest of cytology combined with Xpert® HPV and Anyplex® II HPV28 Detection human papillomavirus (HPV) typing: differential profiles of anal and cervical HPV lesions in HIV-infected patients on antiretroviral therapy.

OBJECTIVES: The aim of the study was to assess the interest to combine cytological examination and human papillomavirus (HPV) typing of anal and cervical Papanicolaou (Pap) smears of HIV-infected patients on combination antiretroviral therapy (cART), to evaluate whether differences in prevalence exist between anal and cervical squamous intraepithelial lesions in patients with high-risk oncogenic HPV infection. METHODS: Anal and/or cervical Pap smears were obtained by anoscopy and/or colposcopy in 238 subjects recruited consecutively in 2015: anal smears were obtained from 48 male and female patients [42 men; 35 men who have sex with men (MSM)] and cervical smears from 190 female patients. Cytological Bethesda classification was coupled with HPV typing. HPV typing was performed, on the same smears, using the Xpert® HPV Assay, which detects only high-risk HPV (hrHPV), and the Anyplex® II HPV28 Detection assay, which detects hrHPV and low-risk (lr) HPV. RESULTS: Our data showed clear-cut differences between the anal and cervical samples. Compared with the cervical samples, the anal samples exhibited (1) more numerous cytological lesions, which were histologically proven; (2) a higher hrHPV infection prevalence; (3) a higher prevalence of multiple hrHPV coinfections whatever HPV typing kit was used; (4) a predominance of HPV16 and HPV18/45 types. Overall, there was an almost perfect agreement between the two HPV typing assays (absolute agreement = 90.3%). CONCLUSIONS: Co-testing consisting of cytology and HPV typing is a useful screening tool in the HIV-infected population on cART. It allows detection of prevalence differences between anal and cervical HPV-related lesions. As recently recommended, anal examination should be regularly performed especially in HIV-infected MSM but also in HIV-infected women with genital hrHPV lesions.

The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

Protein kinase WNK2 inhibits cell proliferation by negatively modulating the activation of MEK1/ERK1/2.

The recently identified subfamily of WNK protein kinases is characterized by a unique sequence variation in the catalytic domain and four related human WNK genes were identified. Here, we describe the cloning and functional analysis of the human family member WNK2. We show that the depletion of endogenous WNK2 expression by RNA interference in human cervical HeLa cancer cells led to the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases but, in contrast to the depletion of WNK1, had no effect on ERK5. Furthermore, expression of a kinase-dead WNK2-K207M mutant also activated ERK1/2 suggesting that WNK2 catalytic activity is required. Depletion of WNK2 expression increased G1/S progression and potentiated the cellular response to low epidermal growth factor concentrations. The molecular mechanism of ERK1/2 activation in WNK2-depleted cells lies downstream of the Raf kinases and involves MEK1 phosphorylation at serine 298 in both HeLa and HT29 colon cancer cells. This modification is linked to the upregulation of MEK1 activity toward ERK1/2. Together, these results provide evidence that WNK2 is involved in the modulation of growth factor-induced cancer cell proliferation through the MEK1/ERK1/2 pathway. The data identify WNK2 as a candidate tumor suppressor gene and suggest a coordinated activity of WNK kinases in the regulation of cell proliferation.

Vasorelaxation induced by prostaglandin E2 in human pulmonary vein: role of the EP4 receptor subtype.

BACKGROUND AND PURPOSE: PGE2 has been shown to induce relaxations in precontracted human pulmonary venous preparations, while in pulmonary arteries this response was not observed. We investigated and characterized the prostanoid receptors which are activated by PGE2 in the human pulmonary veins. EXPERIMENTAL APPROACH: Human pulmonary arteries and veins were cut as rings and set up in organ baths in presence of a TP antagonist. A pharmacological study was performed using selective EP1-4 ligands. The cellular localization of the EP4 receptors by immunohistochemistry and their corresponding transcripts were also investigated in these vessels. KEY RESULTS: PGE2 and the EP4 agonists (L-902688, ONO-AE1-329) induced potent vasodilatation of the human pulmonary vein, pEC50 values: <7.22+/-0.20, 8.06+/-0.12 and 7.80+/-0.09, respectively. These relaxations were inhibited by the EP(4) antagonist GW627368X and not modified in presence of the DP antagonist L-877499. Higher concentrations (>or=1 microM) of the EP2 agonist ONO-AE1-259 induced relaxations of the veins. The EP4 agonists had no effect on the precontracted arteries. Finally, the EP(1) antagonists ONO-8713 and SC-51322 potentiated the relaxation of the veins induced by PGE2. EP4 and EP1 receptors were detected by immunohistochemistry in the veins but not in the arteries. EP4 mRNA accumulation was also greater in the veins when compared with the arterial preparations. CONCLUSIONS AND IMPLICATIONS: Of the 4 EP receptor subtypes, smooth muscle cells in the human pulmonary vein express the EP4 and EP1 receptor subtypes. The relaxations induced by PGE2 in this vessel result from the activation of the EP4 receptor.

A new mRNA splice variant coding for the human EP3-I receptor isoform.

The cellular localization of prostaglandin E2 receptors (EP) and their corresponding transcripts were investigated in human gastric and vascular tissues. A strong staining of the EP3 receptor on the gastric glands, mucous cells, media of the mammary and pulmonary arteries was observed by immunohistochemistry. We identified a new mRNA splice variant of the EP3 gene in human gastric fundic mucosa, mammary artery and pulmonary vessels. This EP3-Ic transcript contains exons 1, 2, 3, 5 and 6 of the EP3 gene and should be translated in the EP3-I isoform. In addition, the EP3-Ib, EP3-II, EP3-III, EP3-IV and EP3-e mRNAs were detected in these tissues.

Altered expression of proteoglycans in E1A-immortalized rat fetal intestinal epithelial cells in culture.

Normal and E1A-immortalized rat fetal intestinal epithelial SLC-11 cells were compared for the characteristics of the 35S-labeled proteoglycans isolated from their cell-associated and secreted fractions. In comparison with control cells in primary culture, immortalized SLC-11 cells: (a) secreted larger amounts of radiolabeled proteoglycans; (b) contained larger amounts of membrane-intercalated proteoglycans, analyzed by hydrophobic affinity chromatography on octyl-Sepharose; (c) produced cell-associated and secreted proteoglycans of smaller hydrodynamic size, assessed by measurement of Kav values; (d) contained a higher percentage of heparan sulfate in the cell-associated proteoglycans, determined by differential susceptibility of glycosaminoglycans to specific glycosaminoglycan lyases; (e) displayed heparan sulfate and chondroitin sulfate with a shorter chain length; and (f) synthesized glycosaminoglycans with a lower degree of sulfation, determined by ion-exchange chromatography. Taken together, these results demonstrate that in E1A-immortalized intestinal epithelial SLC-11 cells, the expression of proteoglycans alters considerably at an early stage of oncogenic transformation.

Increased protein kinase C alpha expression in human colonic Caco-2 cells after insertion of human Ha-ras or polyoma virus middle T oncogenes.

The proteins encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancers. To investigate the mechanism(s) whereby oncogenic p21ras and pp60c-src contribute to malignant transformation of intestine, human colonic Caco-2 cells transfected with an activated (Val 12) human Ha-ras gene (Caco-2-T cells) or Py-MT oncogene, a constitutive activator of pp60c-src tyrosine kinase activity (Caco-2-MT cells), were analyzed for tumorigenicity, protein kinase C (PKC) isoform expression, and PKC activity. As compared with control vector Caco-2-H cells, Caco-2-T and Caco-2-MT cells displayed: (a) an enhanced tumorigenicity in nude mice; (b) a 4-fold increase in the level of PKC-alpha mRNA which was not due to enhanced mRNA stability and was mediated through a PKC-independent pathway since it persisted after PKC depletion; (c) increased PKC-alpha immunoreactive protein content (3-fold), total PKC catalytic activity (3.5-fold), and total cell number of [3H]phorbol-12,13-dibutyrate binding sites (4-fold); and (d) a 1.7-fold higher membrane-bound/total PKC activity ratio together with 1.8- and 1.5-fold increases in [3H]arachidonate- and [3H]myristate-labeled diacylglycerol levels. In conclusion, the tumorigenic progression induced by oncogenic p21ras or the Py-MT/pp60c-src complex in Caco-2 cells is associated with increased PKC-alpha gene transcription and PKC-alpha expression as well as with constitutive PKC activation. These results provide the first evidence that the PKC-alpha gene is a target for the signaling pathways of oncogenically activated p21ras and pp60c-src in human colonic cells. They raise the possibility that PKC-alpha is an effector of these oncoproteins for activation of Caco-2 cell tumorigenic potential.

Down-regulation of carcinoembryonic antigen family member 2 expression is an early event in colorectal tumorigenesis.

Carcinoembryonic antigen gene family member 2 (CGM2), a member of the carcinoembryonic antigen (CEA) family, is expressed in normal colon and rectum but is down-regulated in colorectal adenocarcinomas. In situ hybridization studies demonstrate that CGM2 expression is limited to epithelial cells in the upper third of the crypts. Two other CEA family members, biliary glycoprotein (BGP) and nonspecific cross-reacting antigen (NCA), are similarly expressed, whereas CEA transcripts were found down to the base of the crypts but were less predominant in the upper region. Only low CGM2 and BGP mRNA levels were seen in colorectal tumors. CEA mRNA was expressed at an equivalent level in normal epithelia and in tumor cells, whereas NCA transcript levels were upregulated in tumor cells. Monoclonal antibodies that recognize the CGM2 protein reveal its presence on the apical membranes of epithelial cells in the upper third of the crypts but its absence from colorectal tumors, which do express the CEA and NCA-50/90 proteins. The newly cloned CGM2 3'-untranslated region was used to probe RNAs from adenomas, colorectal tumors at different stages of progression, and liver metastases of colorectal adenocarcinomas. This showed that CGM2 is already down-regulated in adenomas when compared to normal mucosae. The CGM2 expression pattern along with its sequence homology to BGP suggests a similar tumor suppressor function for CGM2.

Loss of sst2 somatostatin receptor gene expression in human pancreatic and colorectal cancer.

Five somatostatin receptor subtypes (sst1 to sst5) have been cloned. We demonstrated previously that sst2 and sst5 mediate the antiproliferative effect of the somatostatin analogues octreotide and vapreotide. Using reverse transcription-PCR, we investigated gene expression of the five receptors in 47 human normal and cancerous tissues or cell lines from pancreatic and colorectal origin. mRNAs of somatostatin receptor subtypes were detected in 98% of samples, with more than two mRNA subtypes being expressed in 55% of cases. sst1, sst4, and sst5 were heterogeneously expressed in both normal and cancerous tissues; sst3 was rarely or not expressed. sst2 was present in normal pancreatic tissues but was absent in exocrine pancreatic carcinomas and their metastases. sst2 mRNAs were detected in normal colon, sporadic polyadenomas, and 50% of Dukes' stage B and 20% of Dukes' stage C carcinomas but were undetectable in Dukes' stage D carcinomas, hepatic metastases, and adenomas from familial adenomatous polyposis. The loss of sst2 expression could represent a growth advantage in these tumors and provide an explanation for the lack of therapeutic effect of somatostatin analogues in such adenocarcinomas. A subtyping of somatostatin receptors should be carried out before considering a somatostatin analogue treatment in patients with colorectal or pancreatic cancer.

Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors.

Rac1 is a member of the Ras superfamily of small GTPases involved in signal transduction pathways that induce the formation of lamellipodia, stimulate cell proliferation and activate the JNK/SAPK protein kinase cascade. Here we describe that amplification by RT-PCR of the entire Rac1 coding sequence from a series of human adult and fetal tissues revealed beside the expected Rac1 cDNA, a variant product which contained additional 57 nucleotides between codons 75 and 76. This variant resulted in an in-frame insertion of 19 new amino acids immediately behind the switch II region, including two potential threonine phosphorylation sites for casein kinase II and protein kinase C. Primers designed within and downstream of the inserted nucleotide sequence allowed isolation of a genomic clone with intronic consensus sequences demonstrating that the insertion corresponds to a novel, yet undescribed exon 3b. This Rac1 splice variant, designated Rac1b, was predominantly identified in skin and epithelial tissues from the intestinal tract. Most notably, the expression of rac1b versus rac1 was found to be elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues. We suggest that the 19 amino acid-insertion following the switch II region may create a novel effector binding site in rac1b, and thus participate in signaling pathways related to the normal or neoplastic growth of the intestinal mucosa.

Evidence for the involvement of the Wnt 2 gene in human colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent cancers in humans. It develops via a multistage process involving alterations of both protooncogenes and tumor suppressor genes. In the present report we determined the level of expression of several Wnt genes in CRC by RT-PCR and direct sequencing. While Wnt-1 was not detectably expressed in any colonic tissues, Wnt-5a gene was efficiently expressed both in nontumorous as well as in colonic tumor tissues. In contrast, the Wnt-2 gene, which was expressed at low levels in normal colon, exhibited overexpression in all tumor tissue samples at the different Dukes' stages of CRC progression, including premalignant polyps and liver metastases. Overexpression of the Wnt-2 gene occurred also in other digestive neoplasms such as gastric and esophageal carcinomas, as well as in diverticulitis associated with stenosis or pseudo-tumor.

Overexpression and amplification of the met/HGF receptor gene during the progression of colorectal cancer.

The c-met oncogene encodes the receptor for hepatocyte growth factor/scatter factor, a potent mitogen for epithelial cells that also promotes cell motility and invasiveness. We have studied the changes of c-met gene expression that occur during the progression of colorectal tumors. Sixteen adenomas, 123 primitive carcinomas, and 25 liver metastases were examined. In several instances it was possible to compare same-patient samples of normal colon mucosa against primary tumor and primary carcinoma against synchronous metastasis. The expression of the c-met gene was increased from 5- to 50-fold in about 50% of tumors, at any stage of progression, and in 70% of liver metastases. Overexpression was associated with amplification of the c-met gene in only 10% of carcinomas, but in 8 of 9 metastases examined. These data suggest that overexpression of the c-met oncogene contributes a selective growth advantage to neoplastic colorectal cells at any stage of tumor progression. Moreover, amplification appears to give a further selective advantage for the acquisition of metastatic potential.

Overexpression of stromelysin-3, BM-40/SPARC, and MET genes in human esophageal carcinoma: implications for prognosis.

Molecular markers can improve staging and predict aggressive clinical behavior in esophageal cancer, thus helping to define appropriate therapeutic protocols and to identify patients who will benefit from surgery. We therefore characterized, by Northern blot and/or immunohistochemistry, the relative expression of three effectors involved in the invasion, angiogenesis, and dissemination of tumor cells in esophageal cancer versus nontumoral mucosae: (a) stromelysin-3 (ST3), a member of the metalloproteinase family; (b) basement membrane 40/secreted protein acidic and rich in cysteine (BM-40/SPARC), an extracellular matrix-associated protein involved in angiogenesis; and (c) the hepatocyte growth factor receptor MET, which triggers the scattering of epithelial cells. Results were analyzed in relation to clinicopathological parameters (cpTNE) including tumor size (T), lymph node status (N), periesophageal tissue invasion (E), disease recurrence, and overall survival. The ST3, BM-40/SPARC, and MET genes were found to be overexpressed in tumor samples compared to control mucosa. BM-40/SPARC and MET mRNA levels were not linked to any one of the cpTNE, indicating that this overexpression occurs at an early stage of neoplastic progression. In contrast, ST3 expression, identified by immunohistochemistry in fibroblastic cells surrounding neoplastic islets, correlated with tumor size and periesophageal tissue invasion. Of the 36 patients studied, those with high ST3 levels had shorter disease-free survival than those with low levels, but there was no relationship between the cpTNE and disease recurrence or survival. Our study demonstrates that ST3, BM-40/SPARC, and MET are involved in different steps of esophageal carcinogenesis and that ST3 overexpression is a marker of aggressive clinical behavior. We conclude that in esophageal cancer, ST3 might help to assess survival and the risk of recurrence after surgical resection.

Ontogenic development of vasoactive intestinal peptide receptors in rat intestinal cells and liver.

Changes in the functional and biochemical characteristics of membrane receptors for vasoactive intestinal peptide (VIP) were evaluated in vitro, using epithelial intestinal cells isolated during rat development, from day 17 of gestation to adulthood. These characteristics included cell cAMP generation, adenylate cyclase and cAMP-dependent phosphodiesterase cAMP-PDE activities, [125I]VIP-binding capacity, and the molecular components of [125I]VIP-binding sites. In 19-day-old fetuses, VIP induced a significant and persistent increase in cAMP production, which lasted for 10 min in intestinal cells. This effect, measured at 37 C in the absence of cAMP-PDE inhibitor, only lasted for 3 min in 5-day-old rats and was undetectable in adult intestine. Addition of the cAMP-PDE inhibitor 3-isobutyl-1-methylxanthine with VIP caused, potentiated, and maintained elevated cAMP levels at the three stages considered. Intestinal cells were more sensitive to VIP in 17- and 19-day-old fetuses (ED50 = 5 and 17 X 10(-11) M VIP, respectively, at 15 and 37 C) than in adult rats (EC50 = 2.7 and 1.6 X 10(-9) M VIP). Adenylate cyclase activity rose 4-fold in fetal intestine and had an apparent Ka of 4 X 10(-10) M VIP. These changes in VIP receptor activity were not observed for PGE2 receptors in developing rat intestinal cells or in the VIP-sensitive adenylate cyclase system prepared from liver of fetuses and adults. They might be due to differences between the molecular components of the intestinal VIP receptor, which were identified here as autoradiographic bands of 64,800 daltons in 19-day-old rat fetuses and 74,600 daltons in adults (P less than 0.01). Alternatively, the changes in VIP receptor activity in 5-day-old rats may result from decreases in the number and affinity of the [125I]VIP-binding sites and increases in the velocity of cAMP-PDE activity. The release of VIP from intestinal nerve endings during fetal and postnatal development and the absorption of VIP from milk might, therefore, modulate the intestinal VIP receptor and its effector systems. Because specific VIP receptors were expressed before the morphological and functional differentiation of intestinal and liver cells, we conclude that their activity is an indicator of their development, and suggest that in rats, this neuropeptide may regulate the maturation and functions of intestine and liver during fetal life.

Vasoactive intestinal peptide receptor activity in human fetal enterocytes.

Functional and specific receptors for vasoactive intestinal peptide (VIP) (determined by their capacity to bind 125I-VIP and activate adenylate cyclase) and cyclic AMP-dependent phosphodiesterase activities were characterized in enterocytes of human fetal small intestine between 18 and 23 weeks of gestation. Half-maximal stimulation of the cyclase and inhibition of 125I-VIP binding in membrane preparations were respectively observed at 1.4 and 5 X 10(-10) M VIP. The peptides structurally related to VIP activated the cyclic AMP generating system at pharmacological doses (10(-7) M and above) in the following order of potency: VIP greater than PHI greater than GRF greater than secretin. Other peptides or test substances, including GIP, pancreatic glucagon, somatostatin-14, gastrin, CCK, neurotensin, pancreatic polypeptide, PYY, substance P, histamine and isoproterenol are inactive in this system, while the ubiquitous adenylate cyclase activators NaF, forskolin and prostaglandins were effective. These results, combined with the appearance of intestinal VIP in nerve fibers at 8 weeks and with the morphological and enzymatic maturation at 9-12 weeks of the intestinal mucosa, indicate that this neuropeptide may regulate either the differentiation or function of enterocytes during the early development of human intestinal mucosa.

Vasoactive intestinal peptide receptor activity and specificity during enterocyte-like differentiation and retrodifferentiation of the human colonic cancerous subclone HT29-18.

Commitment of HT29-18 cells to enterocyte-like differentiation by glucose removal is related to a decreased capacity to generate cAMP after treatment with vasoactive intestinal peptide (VIP), forskolin or sodium fluoride. In contrast, the potency of VIP (EC50 = 1.1 - 1.3 X 10(-10) M) and the pharmacological specificity of the VIP receptor (VIP greater than rh GRF 1-43 greater than PHI greater than secretin) are unchanged during differentiation and retrodifferentiation. These results indicate that disturbances in VIP receptor-post-receptor activity, involving cell surface VIP receptors, membrane and intracellular transducers of hormonal information, occur during enterocyte-like differentiation of the HT29-18 subclone.

Interaction of VIP, PACAP and related peptides in normal and leukemic human monocytes and macrophages.

The activation of the cAMP signaling pathway by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP) and related peptides was studied (i) in normal peripheral human monocytes and THP-1 leukemic human monocytes, (ii) in their derived macrophage counterparts respectively obtained after spontaneous differentiation or retinoic acid (RA) treatment, and (iii) in human bronchoalveolar macrophages. In THP-1 monocytes, PACAP increased basal adenylate cyclase activity 5.3-fold, with an affinity 50-times greater than that of VIP or helodermin (Ka = 3.2 x 10(-11) M VIP), whereas in normal peripheral monocytes, PACAP and VIP exhibited similar affinities and only increased cAMP generation 2-fold (EC50 = 10(-9) M). Spontaneous and RA-induced differentiation into normal and leukemic macrophages induced a progressive loss of cAMP production and regulation of superoxide anion production by VIP and related peptides. The neoplastic transformation in THP-1 monocytes and the deficiencies in the cAMP cascade observed during the terminal differentiation of normal and leukemic human macrophages may relate to a differential genetic expression of the VIP/PACAP receptor subtypes, and alterations in the functional activity of the stimulatory and inhibitory Gs/Gi subunits of adenylate cyclase.

Enterocyte differentiation is compatible with SV40 large T expression and loss of p53 function in human colonic Caco-2 cells. Status of the pRb1 and pRb2 tumor suppressor gene products.

Transfer of the SV40 large-T (LT) oncogene into isolated human and murine intestinal epithelial cells induced alterations of the ultrastructural organization and polarization of the resulting immortalized cell lines. We now demonstrate that the functional expression of the SV40 LT antigen in Caco-2 cells did not alter phenotypic markers of differentiation, including expression of villin, sucrase-isomaltase, brush border and dome formation. As compared to parental cells, the transfected Caco-2 LT9 cells exhibited similar growth curves and no invasive properties in vitro. The major oncogenic function of the SV40 LT antigen in transfected Caco-2 cells is associated with reduced latency times necessary for the manifestation of tumors in athymic nude mice. The Caco-2 cell line contained deleted and mutant p53 alleles (stop codon in position 204) and has no detectable truncated p53 protein by Western blot. Molecular complexes between the SV40 LT antigen and the retinoblastoma-related proteins pRb1 and Rb2 were clearly identified at the different phases of the growth curve. When compared to normal human colonic crypts, Caco-2 cell differentiation is related to partial redistribution of pRb1 into hypophosphorylated, antiproliferative forms. The pRb2 protein is found elevated in a subset of human colorectal tumors and their corresponding liver metastases. We conclude that: (1) Caco-2 cells exert a dominant control against the oncogenic functions of the LT antigen; (2) loss of p53 function is not restrictive for the establishment of polarity and differentiation of the enterocyte lineage; (3) the levels and phosphorylation status of the Rb1 and Rb2 proteins may play important roles in the proliferation and differentiation of normal and neoplastic human colonic mucosa.

Extracellular regulation of cancer invasion: the E-cadherin-catenin and other pathways.

The E-cadherin-catenin complex is pivotal for the regulation of cancer invasion. It not only serves cell-cell adhesion but also transduces signals from the micro-environment to other molecular complexes possibly implicated in invasion. Both functions are disturbed when the extracellular part of E-cadherin is cleaved off. Moreover, upon release into the environment, the E-cadherin fragments may interfere with intact complexes, as indicated by experiments with His-Ala-Val (HAV)-containing peptides that are homologous to parts of the first extracellular domain of E-cadherin. Scatter factor/hepatocyte growth factor (SF/HGF), on binding to its c-met tyrosine kinase receptor, can induce invasion through tyrosine phosphorylation of beta-catenin. SF/HGF-induced invasion is also associated with phosphorylation of pp125FAK, and both invasion and phosphorylation are inhibited by platelet-activating factor (PAF). Activation of the membrane-bound non-receptor tyrosine kinase pp60src can also induce invasion. Signal transduction pathways starting from pp60src include E-cadherin-associated beta-catenin as well as the focal adhesion kinase pp125FAK. Whereas all invasion-inducing pathways implicate phosphoinositide 3-kinase, the PAF pathway seems to be E-cadherin-catenin-independent. We conclude that cancer cell invasion is regulated by paracrine and autocrine factors that are released upon cross-talk with the host cells.

The arotinoid Ro 40-8757 has antiproliferative effects in drug-resistant human colon and breast cancer cell lines in vitro.

We have examined the antiproliferative effects of the arotinoid Ro 40-8757 in 3 drug-resistant human adenocarcinoma cell lines: the colonic cells HT29-5FU and CaCo2, and the mammary cells MCF-7mdr1. Whereas all-trans retinoic acid had no effect at the concentration of 10(-6) M, Ro 40-8757 was found to exert a high antiproliferative action with similar inhibitory potency (IC50) in drug-resistant and parental cell lines (range, 0.06 x 10(-6) to 0.57 x 10(-6) M). We conclude that: (1) thymidylate synthase is not involved in the mechanism of action of Ro 40-8757; (2) the mdr1 gene product does not recognize this retinoic derivative, and (3) Ro 40-8757, alone or in combinations with other cytotoxic drugs, can be very useful in patients with progressive disease after conventional chemotherapy.