CD44 mRNA Expression Research Articles

CD44 is overexpressed in basal-like breast cancers but is not a driver of 11p13 amplification

Overexpression and alternative splicing of CD44 have been implicated in tumour progression. Here we describe the identification of a high level amplification of human 11p13, encompassing the CD44 gene, in primary breast cancers and cell lines and test whether CD44 acts as the driver of this amplicon. aCGH analysis revealed 11p13 amplification in 3% (3/100) of primary breast carcinomas and in two cell lines. The minimal region of amplification was 34.38-37.62 Mb. Amplification was confirmed by dual-colour FISH in these cell lines and further validated by CISH in an independent tumour cohort. CD44 expression in primary breast cancers was significantly associated with features of basal-like breast cancer. Detection of CD44 expression in breast cancer cell lines confirmed moderate to high expression in basal-like cell lines and minimal expression in luminal cell lines. In both, primary breast cancers and cell lines, 11p13 amplification was associated with high levels of CD44 mRNA expression. CD44 alternative splicing was detected in four of nine cell lines and in tumour samples, irrespective of the amplification status. RNAi mediated knock down of CD44 failed to reveal an increased dependence on CD44 expression for proliferation or survival in amplified cell lines. Given that expression of CD44 is not an absolute requirement for the survival of cells harbouring CD44 gene amplification, CD44 is unlikely to be a driver of the 11p13 amplicon.

CD133 expression in different stages of gastric adenocarcinoma

Sir, With great interest, we have studied the work by Smith et al (2008) that describes presence of CD133 receptors on gastrointestinal cancer cells and states that an antibody-drug conjugate against this receptor led to delayed growth response in gastric cancer cell lines as well as in mice. The work supports the hypothesis that CD133+ cells act as tumorigenic cancer cells. In reaction to the published work, we have investigated that quantitative CD133 expression in patients with different stages of gastric cancer compared with normal mucosa or chronically inflamed tissue. Interestingly, this expression was reduced in cancer patients and was significantly decreased in patients with more advanced stages of cancer (see Figure 1). These results question the role of CD133 as a marker for tumorigenic gastric cancer cells or question the hypothesis of the potential of such stem cells. Figure 1 (A) CD133 mRNA expression in human gastric tissue samples from 92 patients. mRNA copy numbers were normalised to 106 GAPDH copies. The histological presentations are normal stomach without H. pylori infection or inflammation (n=20), H. pylori infection ... In contrast to the work by Smith et al (2008) we determined mRNA levels by RT–PCR, as we were not able to establish a specific antibody binding. This may be because of the scarcity of such tumorigenic cancer cells. In colon cancer, only 2.5% of all cancer cells were found to express CD133 receptors (Ricci-Vitiani et al, 2007). If CD133+ cancer cells act as tumorigenic cells although being able to divide, our data question the function of such cells as they were actually reduced in patients with more advanced stages and distant metastasis; in our study, the greatest CD133 expression was found in mucosal stages that are known to have lymph node metastasis only in very rare cases (Gotoda et al, 2000). Alternatively, CD133 receptors may be expressed on other cells than tumour cells. Our data point towards a potential role of CD133 in angiogenesis as VEGF-R2 receptors were closely related to CD133 expression in our collective (correlation coefficient: 0.306; P=0.008). Endothelial progenitor cells (EPCs), a subgroup of bone marrow-derived stem cells, were shown to express CD133 receptors together with VEGFR-2, CD34 and VE-cadherin (Peichev et al, 2000). Here, CD133 expression was reduced in advanced stages of cancer, paralleling VEGF-R2 expression and microvessel density. Thus, it seems to be that the decrease of CD133 mRNA in advanced stages of gastric cancer reflects reduced angiogenesis and simultaneously reduced presence of endothelial precursor cells. Overall, the earlier observation of a functional importance of CD133 may be related to the inhibition of angiogenesis and vessel formation, but not directly to a functional role of these receptors in cell proliferation of malignant tumour cells.

Sinomenine influences capacity for invasion and migration in activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, and CD147

The aim of this study was to investigate the mechanism of the effects of Sinomenine (SIN) on the invasion and migration ability of activated human monocytic THP-1 cells (A-THP-1). Sinomenine is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum. Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Cells were treated with different concentrations of SIN. The invasion and migration ability of cells was tested by in vitro transwell assays. The levels of CD147 and MMPs were evaluated by flow cytometric analysis and zymographic analysis, respectively. The mRNA expression of CD147, MMP-2, and MMP-9 was measured by RT-PCR. The invasion and migration ability of A-THP-1 cells was significantly inhibited by SIN in a concentration-dependent fashion; at the same time, the levels of CD147, MMP-2, and MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 mmol/L and 1.00 mmol/L (P<0.01). A possible mechanism of the inhibitory effect of SIN on cell invasion and migration ability is repression of the expression of MMP-2 and MMP-9, which strongly correlates with the inhibition of CD147 activity.

Comparison of cyclooxygenase-2 and CD44 mRNA expression in colorectal cancer and its relevance for prognosis

This study evaluated CD44 and COX-2 expression in colorectal cancer (CRC) and analyzed its relationship with the clinicopathological characteristics. The prognostic impact on patient survival was compared between the two proteins. CD44 and COX-2 mRNA levels in 42 primary CRCs were analyzed using quantitative real-time PCR, with normalization relative to GAPDH. The cycle threshold (Ct) values were measured, and results are expressed as the Ct ratios of CD44 or COX-2 to GAPDH. The COX-2 Ct ratio was much lower in cases of lymphovascular invasion by the tumor than for no invasion (P = 0.004). During follow-up for a median of 40 months, there was no significant difference in the median CD44 Ct ratio between survivors and non-survivors (P = 0.362), whereas the COX-2 Ct ratio was significantly associated with survival at the time of data analysis (P = 0.042). The survival of colorectal cancer patients with a high COX-2 Ct ratio was significantly longer than that of patients with a low COX-2 Ct ratio (P = 0.048). This study suggests that COX-2 expression has a more significant impact than CD44 expression on the survival of CRC patients. Further studies are needed to resolve these issues with a large sample size.

Enhanced CD24 expression in endometrial carcinoma and its expression pattern in normal and hyperplastic endometrium.

CD24 is known to be an important diagnostic and prognostic marker of several major cancers affecting females. We aimed to determine CD24 expression in normal, hyperplastic, and carcinomatous endometrium and its correlation with estrogen and progesterone receptor expression. A total of 271 cases including 62 normal/atrophic endometrium cases (47/15), 127 endometrial hyperplasia cases (51/52/24, simple/complex/atypical hyperplasia), and 82 endometrial carcinoma cases were immunohistochemically analyzed by using anti-CD24, ER, and PR antibodies that were embedded on paraffin blocks. Next, we assessed the CD24 mRNA expression in these tissues by using RT-PCR. In the normal endometrium, cyclic expression of membranous CD24 was detected during the regular menstrual cycle, i.e., down-regulation in the proliferative phase and up-regulation in the secretory phase. CD24 expression was very infrequent and weak in the atrophic endometrium. In hyperplasias and carcinomas, the expression of both membranous and cytoplasmic CD24 was found to be sharply reduced in the hyperplastic lesions and significantly enhanced in the carcinomas. In the case of carcinomas, high CD24 expression showed significant correlation with high-grade (G2 and 3) (P<0.05). In addition, an inverse correlation was apparent between CD24 and the estrogen and progesterone receptor expressions in normal and diseased endometrium. In conclusion, we demonstrated that CD24 was expressed in a cyclic pattern in the normal endometrium, and its expression was enhanced in case of endometrial carcinoma. These results suggest that CD24 may be involved in tumor progression and can be a useful diagnostic biomarker.

Expression of CD147 on phorbol-12-myris-tate-13-acetate (PMA)-treated U937 cells differentiating into foam cells

Monocytes, macrophages, and foam cells expressing CD147 can stimulate the production of several matrix metalloproteinases (MMPs) associated with the development of atherosclerosis. We defined the CD147 expression profile and examined the correlation between foam cell development and MMP-2, -9 expressions. Foam cells were derived from U937-stimulated macrophages using various concentrations of oxidized low-density lipoprotein (ox-LDL). PMA-stimulated U937 cells had a 4- to 5-fold increase in CD147 mRNA compared to undifferentiated monocytes and membrane-associated (mCD147) on foam cells decreased in response to ox-LDL in a dose-dependent manner compared to untreated macrophages. In contrast, ox-LDL treatment increased the levels of soluble CD147 (sCD147) and MMP-2, -9 in a dose-dependent manner. Our data suggested that monocyte differentiation up-regulated CD147 expression and lipid enrichment of foam cells had no effect on CD147 mRNA expression. Lipid loading in macrophages reduced mCD147 expression while increasing the levels of MMP-2, -9 and sCD147 in supernatants.

Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Genetic Modulation of CD44 Expression by Intragraft Fibroblasts

This study investigated the genetic composition and the functional implication of CD44 species expressed by intragraft fibroblasts. An LEW-to-F344 heart transplant model of chronic rejection was used. Intragraft fibroblasts recovered from the chronically rejecting allografts displayed a 4.5-fold increase in expression of CD44 mRNA when compared with that of the fibroblasts isolated from non-rejecting heart allografts (P < 0.01). The intragraft fibroblasts preferentially expressed CD44 variant isoforms containing v1 exon transcript. Automated nucleotide sequence analysis revealed that the majority (90.12%) of the CD44 v1 isoforms expressed by the rejecting graft fibroblasts were encoded by a mutated CD44 mRNA, which contained two point mutations and a codon deletion in the v1 coding region. Histochemistry demonstrated a massive deposition of extracellular HA in the rejecting heart allografts. Hyaluronic acid (HA) was able to promote in vitro fibroblast adhesion, migration in a CD44-dependent manner, and survival in a serum-free culture condition. The study concludes that up-regulation of CD44 v1 isoforms expressed by the intragraft fibroblasts is associated with an increase in the deposition of extracellular HA, the principal ligand for CD44, in the allografts, suggesting that CD44-HA interaction plays an important role in regulating fibroblast recruitment and growth in allografts developing chronic rejection.

A fraction of methylene chloride from Geum japonicum Thunberg inhibits tumor metastatic and angiogenic potential

The plant Geum japonicum Thunberg (GjT) has been used as a diuretic in traditional medicine. Herein, we report that the GjT extract blocks both the spread of human umbilical vein endothelial cells (HUVECs) on matrigel and the migration of B16 cells. We used various assays to test for cell attachment, spreading, wound healing and angiogenesis. A reverse transcription-polymerase chain reaction (RT-PCR) and a mitogen-activated protein kinase (MAPK) assay were also carried out for the mechanistic study of GjT. Our results showed that a fraction of methylene chloride fraction from GjT inhibited B16 cells during cell attachment and migration and suppressed tube formation in a dose-dependent manner. An RT-PCR analysis showed that the methylene chloride extract decreased the mRNA expression of CD44 and TIMP-2. A Western blot analysis of the phosphorylation of MAPK kinases (ERK, JNK and p38) showed that the GjT fraction increased the expression of phospho-JNK, suggesting that GjT has the potential to alleviate metastatic and angiogenic activity, via a phospho-JNK signaling pathway.

Anti-tumor activity of Gastrodia elata Blume is closely associated with a GTP-Ras-dependent pathway

Gastrodia elata Blume (GEB) is an important medicinal plant in Korea. In order to confirm the anti-tumor activities of GEB extracts, we carried out various in vitro anti-tumor assays, including a wound assay and an invasion assay using an ethyl ether extract of GEB. The results showed that the GEB extract exhibits potent anti-tumor activity in vitro in a dose-dependent manner. The expression of CD44, cdc42, Timp-2 or RhoA mRNA did not change by GEB treatment, compared to that of the control. GTP-Ras, an active form of a G-coupled protein family, however, is associated with the anti-tumor activity of GEB extracts. We examined various molecular markers related to metastasis by reverse transcriptase-polymerase chain reaction with the extract of GEB-treated B16 cells. There was an increase in GTP-Ras expression by the Gastrodia elata Blume extract. Together, these results suggest that the Gastrodia elata Blume extract could have potential in alleviating tumorigenesis, by a GTP-Ras-dependent pathway; although the precise molecular mechanisms are still being examined.

Phase I Pharmacokinetic and Pharmacodynamic Study of the Oral Protein Kinase C β-Inhibitor Enzastaurin in Combination with Gemcitabine and Cisplatin in Patients with Advanced Cancer

Enzastaurin targets the protein kinase C and phosphatidylinositol 3-kinase/AKT pathways to reduce tumor angiogenesis and cell proliferation and to induce cell death. A phase I trial was conducted to evaluate the feasibility of combining enzastaurin with gemcitabine and cisplatin. Patients with advanced cancer received a 14-day lead-in treatment with oral enzastaurin followed by subsequent 21-day cycles of daily enzastaurin, gemcitabine on days 1 and 8, and cisplatin on day 1. Enzastaurin doses were escalated between 350 mg once daily to 500 mg twice daily, whereas gemcitabine doses were either 1,000 or 1,250 mg/m(2) and cisplatin doses were either 60 or 75 mg/m(2). Circulating endothelial cell numbers and CD146 and CD133 mRNA expression were evaluated as pharmacodynamic markers. Thirty-three patients (median age, 58 years) were enrolled in seven dose levels. The maximum tolerated dose was not identified. Two dose-limiting toxicities (grade 2 QT interval corrected for heart rate prolongation and grade 3 fatigue) were reported. Other toxicities included grade 3/4 neutropenia (3 of 6 patients), thrombocytopenia (1 of 6 patients), grade 3 leukopenia (2 patients), and fatigue (5 patients). Enzastaurin twice daily (> or =250 mg) resulted in more discontinuations and low-grade toxicities. In the combination, enzastaurin exposures decreased slightly but remained above the target of 1,400 nmol/L, whereas gemcitabine/cisplatin exposures were unaltered. Three patients (9.1%) had partial responses and 13 (39.4%) had stable disease. Measurement of circulating endothelial cell numbers and CD146 and CD133 mRNA expression did not contribute to decision-making on dose escalation. Recommended phase II dose is 500 mg enzastaurin once daily, 1,250 mg/m(2) gemcitabine, and 75 mg/m(2) cisplatin. This regimen is well tolerated with no significant alterations in the pharmacokinetic variables of any drug.

Elevated stem cell marker CD133 mRNA in peripheral blood predicts colon cancer recurrence

10504 Background: CD133 is a specific stem cell marker that enriches cancer stem cells of many tumor types including colon cancer as well as circulating endothelial progenitors (CEP). CEP is vital in postnatal angiogenesis and elevated CEP is a proven favoable prognositic marker for heart disease but a potentially poor prognostic marker for cancer. We examined whether elevated CD133 mRNA expression levels in peripheral blood mononuclear cells (PBMC) predict recurrence in colon cancer patients. Methods: We developed and validated a semi-quantitative real-time RT-PCR to quantify CD133 mRNA levels relative to GADPH mRNA. Sixty-six colon cancer patients were enrolled between February 2002 and December 2003. The protocol excluded patients with history of cardiac disease or surgery &lt; 4 weeks from the enrollment and were followed for recurrence for a median 30 months. A central statistician performed multivariate unconditional logistics regression analysis. Results: Among the patients without recurrence, 93% had a CD133 mRNA level &lt; 4.79, whereas 7% had a CD133 mRNA value ≥ 4.79 (p = 0.029). Among patients with a CD133 mRNA value ≥ 4.79, 85% had experienced recurrence compared to 15% of the patient who had no recurrence (p = 0.03). Elevated CD133 mRNA levels at a cut-off point ≥ 4.79 versus &lt; 4.79 were associated with an odd ratio of 22.6 for recurrence (95% CI, 1.7–291.2; p = 0.02); in comparison, the odds ratio for recurrence was 17.2 (95% CI, 1.8–164; p = 0.01) for stage IV patients versus stage I-III patients. No other predictive variables for recurrence were identified including age, race, sex, tumor differentiation, smoking, and diabetes etc. We also observed an trend of association with elevated carcinoma embryonic antigen (CEA) levels (p = 0.03, one sided) and a decreased survival (p = 0.035, one sided) with elevated CD133 mRNA level at a cutoff point ≥ 4.79. Conclusions: Elevated CD133 mRNA levels at a cutoff ≥ 4.79 in PBMC predict colon cancer recurrence independent of stage IV disease. The current assay has certain advantages over flow cytometry for wider clinical application. CD133+ cells measured by CD133 mRNA may contain both CEP and cancer stem cells, leading to increased risks of recurrence. No significant financial relationships to disclose.

445: Preferential expression of CD44 v1 isoforms by intragraft fibroblasts from cardiac allografts with chronic rejection

Purpose: CD44 is a transmembrane glycoprotein primarily involved in cell-cell interactions and cell migration. This study investigated the genetic composition and the functional implication of CD44 species expressed by intragraft fibroblasts (IF). Methods and Materials: Intragraft fibroblasts recovered from the chronically rejecting heart allografts of rats were examined for CD44 gene expression using quantitative real time PCR. CD44 specific cDNA libraries were constructed and selected CD44 clones were analyzed in automate nucleotide sequencing. The role of CD44/hyaluronan interaction was studied using in vitro migration and proliferation assays. Results: IF displayed a 4.5-fold increase in expression of CD44 mRNA when compared with that of the fibroblasts isolated from non-rejecting heart allografts (p 0.01). The IF preferentially expressed CD44 variant isoforms containing v1 exon transcript. Automate nucleotide sequence analysis revealed that the majority (90.12%) of the CD44 v1 isoforms expressed by the rejecting graft fibroblasts were encoded by a mutated CD44 mRNA, which contained 2 point mutations and a codon deletion in the v1 coding region. Histochemistry demonstrated a massive deposition of extracellular hyaluronan in the rejecting heart allografts. Hyaluronan promoted in vitro fibroblast adhesion, migration in a CD44 dependent manner, and survival in a serum-free culture condition. Conclusions: A significant increase in CD44 expression by the rejecting graft fibroblasts was coupled with augmentation of extracellular hyaluronan synthesis in the heart allografts, indicating that CD44-HA interaction is critically involved in the pathogenesis of graft fibrosis. CD44v1 isoforms may play a role, in a way similar to other documented v exons such as v3 or v6, to provide additional signaling for enhancing or suppressing the biological activities of intragraft fibroblasts. CD44 antagonism may affect multiple cellular activities mediated by the CD44-HA pathway, leading to alleviation of allograft fibrosis and chronic inflammation.

Fra-1 governs cell migration via modulation of CD44 expression in human mesotheliomas

Silencing of Fra-1, a component of the dimeric transcription factor, activator protein-1 (AP-1), inhibits mRNA expression of c-met and cd44 in rat mesothelioma cells and is causally linked to maintenance of the transformed phenotype. However, the mechanisms of Fra-1 regulation and Fra-1 regulated gene expression in human malignant mesothelioma (MM) are unclear. We first show in a panel of human MM cells that Fra-1 mRNA expression in MM is complex and regulated by extracellular signal-regulated kinase (ERK1, ERK2), Src, and phosphatidyl-inositol-3-kinase (PI3K) pathways in a tumor-specific fashion. Cell lines with PI3K-dependent Fra-1 expression were SV40 positive and expressed the lowest basal Fra-1 levels. Levels of Fra-1 expression correlated with amounts of CD44 expression that were greater in simian virus 40 negative (SV40-) MM cells. Using dominant negative (dn), short hairpin (sh) and small interference (si) RNA constructs, we next demonstrate that expression of CD44, the principal hyaluronic receptor in MMs, correlates with Fra-expression in both simian virus 40 positive (SV40+) and SV40- MMs. Moreover, both Fra-1 and CD44 expression are linked to cell migration in SV40- MM cells. Lastly, in contrast to normal lung tissue, tissue microarrays revealed that Fra-1 was expressed in 33 of 34 human MMs, and that all CD44+ tumors were SV40-. These results suggest that Fra-1 is associated with cell migration in human MMs and that Fra-1 modulation of CD44 may govern migration of selected MMs.

DNA/Lipid Complex Incorporated with Fibronectin to Cell Adhesion Enhances Transfection Efficiency in Prostate Cancer Cells and Xenografts

Previously we have described the development and applications of lipid-based nanoparticles for gene delivery vector. In an attempt to improve transfection efficiency using the cell adhesion of extracellular matrix (ECM) to DNA/lipid complex (nanoplex), the mRNA expression of integrin alpha2beta1 and CD44 in prostate cancer cells was detected as adhesion molecules for fibronectin (Fn), collagen I (Col) and laminin (Lam) using a commercially available cDNA array (GEArray) system. These ECM proteins could enhance DNA transfection activity in cells when coated on the nanoplex. Among the ECM proteins, Fn-coating nanoplexes significantly increased transfection activity 2-fold in prostate cancer PC-3 cells, and exhibited higher DNA transfection activities to PC-3 xenografts, compared with commercially available cationic polymer in vivo jetPEI. These results indicated that Fn-coating nanoplexes could facilitate efficient transfection of prostate tumor cells.

Progenitor Marker CD133 mRNA Is Elevated in Peripheral Blood of Cancer Patients with Bone Metastases

We examined whether RNA expression of CD133, a surface molecule expressed on progenitors from hematopoietic and endothelial lineages, and CD146, a pan-endothelial marker, are increased in the blood of cancer patients and whether these factors correlate with patient characteristics and are predictive factors of survival. We developed a real-time quantification method (nuclear acid sequence-based amplification) to determine expression of CD146 and CD133 mRNA in the peripheral blood mononuclear cells of 131 progressive cancer patients, 37 healthy volunteers, and 5 patients who received granulocyte colony-stimulating factor. Overall survival and other clinicopathologic variables were obtained. Cox proportional hazards studies were done. We show that patients with metastatic disease have a significant increase in CD133 mRNA (P = 0.03), specifically patients with bone metastasis (P < 0.001). Cancer patients with high CD133 mRNA expression, using a defined cutoff value, show a decreased survival compared with patients with low or undetectable CD133 expression (21% versus 45% cumulative survival, respectively, after 20 months; P = 0.01). Among patients with metastasis to the bone, cumulative survival was 22%, compared with 61% for patients with high or low CD133 levels (P = 0.004). Multivariate analysis showed that CD133 expression is an independent predictor for overall survival in patients with bone metastases. CD146 mRNA was not increased in patients with cancer, nor did it correlate with clinical variables or survival. CD133, but not CD146, mRNA expression is increased in cancer patients with metastatic disease, specifically with bone metastasis. In addition, CD133 mRNA expression seems to be an independent prognostic factor for overall survival.

Endothelial progenitor marker CD133 mRNA expression in peripheral blood mononuclear cells predicts outcome of cancer patients

10087 Background: We examined whether expression of CD133, a surface molecule expressed on progenitors of hematopoietic and endothelial lineages, is increased in the peripheral blood mononuclear cells (PBMC’s) of cancer patients. Furthermore we correlated CD133 mRNA expression with tumor prognostication factors and overall survival. Methods: PBMC’s were isolated from the blood of 130 advanced cancer patients (43 renal cell carcinoma, 27 colorectal cancer, 34 prostate cancer, 27 head and neck cancer). Clinicopathological parameters were obtained. Median follow-up time was 16 months after inclusion (range 3–33 months). For the quantification of CD133 mRNA we used a real-time detection and quantification method based on nucleic acid sequence-based amplification (NASBA). Also U1A DNA was quantified as internal control for input and isolation. CD133 mRNA copy numbers were expressed as copies per 10.000 cells, as determined by U1A DNA. Results: Patients with metastasized disease have a significant increase in CD133 mRNA as compared to patients without metastasis. This increase is strongest in patients with bone metastasis. Subgroup analysis in patients with bone involvement show that CD133 mRNA also correlates with PSA (n=34)and inversely with hemoglobin (n=50). CD133 mRNA expression is an independent predictor for overall survival in Cox’s multivariate analysis (variables in the model included age, hemoglobin, metastasis, previous treatment and CD133 as categorical variable). A CD133 mRNA cut-off value was established by Receiver Operating Characteristic analysis of CD133 expression in relationship to survival (sensitivity 64% and specificity 71%). Cancer patients with more than 200 copies of CD133 mRNA, demonstrate a decreased survival compared to patients with lower CD133 expression (median survival 8 and 15.4 months, respectively). In patients with bone metastasis the median survival of high and low expressers is 6.3 months and 21.9 months, respectively. Conclusions: mRNA expression of stem-cell marker CD133 is increased in cancer patients with metastasized disease and an independent prognostic factor for overall survival. Increased CD133 mRNA expression may reflect an activated angiogenic state in these cancer patients. [Table: see text]

Quantitative real time-PCR of CD133 mRNA: A potential surrogate angiogenic marker of response for patients with metastatic sarcoma

20034 Background: CD133 antigen is a specific surface marker for circulating endothelial progenitor cells (CEPs), pivotal in post-natal angiogenesis. We hypothesize that changes in levels of the CD133 mRNA expression and vascular endothelial growth factor (VEGF) may correlate with tumor response. Methods: After informed consent, we obtained 48 peripheral blood samples from patients with metastatic sarcoma including gastrointestinal stroma tumors (GIST). There were 16-paired samples before and after treatment including chemotherapy, or surgery, or imatinib. Of 24 patients with metastatic GIST enrolled, seven were paired samples. We measured CD133 mRNA levels by method (Lin E et al AACR 2003# 5392) and VEGF levels by ELISA (Genzyme, MA). The measurements were done in duplicates in two experiments. Results: The mean CD133 levels and VEGF levels before and after treatment was in Table 1 . The treatment resulted in significant reduction of CD133 mRNA expression (p = 0.035) as well as the level of VEGF (p = 0.014). Three patients experienced increase in CD133 expression had progressive disease. Among the paired GIST patients, there was a trend of increased CD133 mRNA expression levels in patients with progressive disease, death, or tumor recurrence. CD133 mRNA levels appeared elevated among the imatinib naïve patients and imatinib appeared to reset CD133 mRNA levels among the responding GIST patients to that of healthy volunteers. Conclusion: CD133 mRNA expression levels in sarcoma patients measured by real time-PCR assay appeared to correlate with tumor response. Unpublished independent work with NASBA assay platform in other solid tumors supported our findings ( www.primagen.com ). Further work is needed to reduce assay variability, and to determine the sensitivity and specificity of assay comparing to flow cytometry and to test assay’s applicability in the antiangiogenic therapy of cancer and the angiogenic therapy of cardiovascular diseases. [Table: see text] No significant financial relationships to disclose.

Expression of CD44 in rat hepatic progenitor cells

Small hepatocytes (SHs) are hepatic progenitor cells, but the phenotypical difference between SHs and mature hepatocytes (MHs) has never been demonstrated. The profile of gene expression was examined to clarify the difference between SHs and MHs by using a DNA microarray. Genes that were specifically expressed in SHs were identified and RT-PCR analysis of them was performed. Immunocytochemistry for CD44 standard form (CD44s) and variant form 6 (CD44v6) was performed using cultured SHs and the d-galactosamine (GalN)-injured rat liver. From the GalN-treated liver, CD44s+ cells were obtained by sorting and RT-PCR analysis was performed. Analysis using the DNA microarray and RT-PCR of them revealed restricted expression of CD44s and CD44v6 in SHs. In culture, CD44s appeared at day 3 and increased with the proliferation of SHs. CD44v6 expression was delayed compared to that of CD44s. With GalN-administration, CD44+ hepatocytes appeared around periportal areas at days 3 and 4 and then decreased. Sorted CD44s+ cells could form colonies and possessed hepatic markers. CD44 is a specific marker of SHs. The expression of CD44 mRNA and protein is restricted to SHs, and is up-regulated at the time when SHs start to proliferate both in vitro and in vivo.

3′ Distal Regulatory Elements Required for Human CD34 Expression in Transgenic Mice.

CD34 is the best-defined human hematopoietic stem cell (HSC) marker, however the regulation of its gene expression is still largely unknown. Therefore, unraveling the elements that regulate human CD34 expression would be an invaluable tool for a broad range of studies, including the establishment of models of leukemia in mice, which require targeting of the transgene to stem and/or early progenitor cells. Moreover, identification of such regulatory elements will provide important insights into the transcriptional agenda of stem and progenitor cells and most importantly will prove useful for gene therapy protocols. Studies from our laboratory demonstrated that human CD34 transgenes are expressed in murine repopulating HSCs, which resembles the expression of the CD34 gene in human hematopoiesis, thus indicating the mouse model as an excellent way to study the expression of human CD34. Using P1 derived artificial chromosome (PAC) clones encompassing the human CD34 gene to generate transgenic mice, we showed that 90kb of upstream and 26kb of downstream flanking sequences were capable of regulating human CD34 expression in murine transgenic lines. Successive deletions of this larger construct were then performed to identify the important control regions. Deletion of the 5′ region from −90kb to −18kb did not result in any loss of activity. PAC54A19, a clone extending from −18kb to +26kb, expressed RNA in various tissues in a manner similar to that of larger fragments. In contrast, deletions creating a construct spanning from −10kb to +17kb led to complete loss of expression in transgenic animals, indicating that critical distal elements are located between −18kb to −10kb and/or +17kb to +26kb. In order to facilitate identification of important regulatory elements present in the upstream (−18kb to −10 kb) and/or downstream (+17kb to +26kb) regions of human CD34, we created further deletions of PAC54A19 using rare-cutting restriction enzymes, and studied the effects of the deletions on human CD34 expression in transgenic mice. Interestingly, we did not detect any human CD34 mRNA and protein expression in bone marrow and HSCs from transgenic mice carrying a construct spanning from −18kb to +17.4kb. In contrast, we observed expression of human CD34 transcripts in the bone marrow of transgenic mice containing a PAC spanning from −12.8kb to +26kb. Furthermore, HSCs from this latter group of mice presented the human CD34 antigen on their surface, as detected by FACS. Taken together, these data are highly suggestive that critical cis regulatory element(s) required to drive human CD34 in vivo expression are located in a 8.6kb fragment placed between +17.4kb and +26kb downstream of the human CD34 gene. Our current efforts focus on identifying the element(s) within the 8.6kb 3′ region that might be required to achieve human CD34 expression in HSCs.