Symmetric division of cancer stem cells--a key mechanism in tumor growth that should be targeted in future therapeutic approaches.

As cancer stem cells (SCs) drive tumor growth, it is only through the elimination of those cancer SCs that a pharmacologic cure can be attained. To study ways to develop drugs that target cancer SC, we investigated changes in cellular mechanisms and kinetics that occur in SC populations during colorectal cancer (CRC) development. We used computer modeling to determine which changes could give rise to exponential increases in both SC and non-SC populations in CRC. Our results show that the only mechanism that can explain how these subpopulations increase exponentially in CRC development involves an increase in symmetric SC cell division. This finding suggests that any systemic therapies designed to effectively treat CRC and other cancers must act to control or eliminate symmetrical cancer SC division in tumors, while minimally affecting normal SC division in non-tumor tissues.

Development of cross-resistance to tamoxifen in raloxifene-treated breast carcinoma cells.

Selective estrogen receptor modifiers (SERMs) are used chronically in the treatment of breast cancer and osteoporosis but some patients become resistant, at which point second-line SERMs are considered as options. Because the use of SERMs is increasing and breast cancer is so common, we tested the hypothesis that treatment with SERMs can induce cross-resistance to other SERMs. We used three cultured breast carcinoma cell lines (MCF-7, ZR-75-1, and T47D) which are estrogen-receptor-positive (ER+) and are prone to developing resistance to hormonal treatment. Cell lines were exposed to increasing doses of raloxifene. Raloxifene-resistant clones were selected and tested for cross-resistance to tamoxifen. Compared to untreated cells, raloxifene-resistant clones showed an increased IC50 (reduced potency) of about 15,000-fold with no apparent change in maximal inhibition of cell growth. These same raloxifene-resistant clones were also about 15-fold more resistant to the growth-inhibiting effects of tamoxifen. While the resistance to tamoxifen is considerably less marked (1000-fold less), it is large enough to raise the question as to whether patients who become resistant to raloxifene will benefit by switching to tamoxifen or vice versa.

Computer modeling implicates stem cell overproduction in colon cancer initiation.

On the basis of our investigation of the premalignant crypt phenotype in familial adenomatous polyposis patients, the hypothesis is developed that tumor initiation in the colon is caused by crypt stem cell overproduction. A novel kinetic model for the colonic crypt was used to investigate how the earliest tissue abnormality (altered crypt labeling index) arises in these patients who have a mutant APC genotype. Only an increase in crypt stem cell number, not changes in the rate of cell cycle proliferation, differentiation, or apoptosis of the non-stem cell population, simulated this abnormality. This suggests that APC regulates the number of stem cells in the colonic crypt and when the cells become mutant, an expansion of the crypt stem cell population results.

Evidence that APC regulates survivin expression: a possible mechanism contributing to the stem cell origin of colon cancer.

Because colorectal cancers (CRCs) frequently display APC mutation, inhibition of apoptosis, and increased expression of the antiapoptotic protein survivin, we hypothesized that APC mutation inhibits apoptosis by allowing constitutive survivin expression. Using HT-29 CRC cell lines having inducible wild-type APC (wt-APC) or transfected dominant-negative TCF-4, we show that wt-APC down-regulates survivin expression via APC/beta-catenin/TCF-4 signaling. Using normal colonic epithelium, we found survivin by immunostaining/reverse transcription-PCR to be preferentially expressed in the lower crypt (which inversely correlates with wt-APC's expression pattern). Thus, wt-APC, by progressively decreasing survivin and increasing apoptosis from crypt bottom to top, may limit the population size of stem cells and other proliferative cells in the lower crypt; mutant APC may allow expansion of these populations, thereby initiating tumorigenesis.

Postoperative management of local colorectal cancer: therapy and surveillance.

Adjuvant therapy is widely recommended for stage III colon cancer and stages II and III rectal cancer. Although fluorouracil-based regimens are standard, newer agents either alone or in combination may improve response rates. Although nearly all patients enter a postoperative surveillance program after surgical resection, the clinical effectiveness of such surveillance, which is not standardized, is questionable. Critical review of the use of different components (laboratory, radiographic, and endoscopic) of these programs finds little support for intensive surveillance.

Colorectal cancer staging and adjuvant chemotherapy.

Colorectal cancer is a significant cause of morbidity and mortality in Western populations. The standard of care for staging patients with colorectal cancer to determine prognosis and identify patients who will receive adjuvant therapy continues to be histopathology of regional lymph nodes. However, the significant variability in survival within each staging category likely reflects the heterogeneity of detecting micrometastatic disease employing this technique. Novel molecular markers of micrometastases currently in development will permit more accurate staging of patients with colorectal cancer. These advances in staging will distinguish patients who will maximally benefit from adjuvant therapy from those who have an especially good prognosis in whom chemotherapy can be avoided. In addition, new adjuvant chemotherapeutic agents, novel combinations of those agents and creative dosing schedules currently being investigated will offer considerable advantages with respect to ease of administration, safety and tolerability, quality of life and efficacy. Ultimately, it is anticipated that advances in molecular diagnostics will define unique biochemical characteristics of patients' tumours, permitting individualization of chemotherapeutic regimens employing novel agents that specifically exploit those characteristics.

Tumor-specific expression of anti-mdr1 ribozyme selectively restores chemosensitivity in multidrug-resistant colon-adenocarcinoma cells.

P-glycoprotein (Pgp)-conferred multidrug resistance (MDR) is expressed in cancer and in normal colon tissues and has important physiological functions. In order to selectively reverse MDR in malignant tissue without disrupting the function of normal colonocytes, a retroviral vector (pCEAMR) containing anti-mdr1 ribozyme coupled to the carcino-embryonic-antigen (CEA) promoter was constructed and introduced into resistant colon-cancer cells (SW1116R) that produce CEA and into control resistant cells (HeLaK) that do not produce CEA. Anti-mdr1 ribozyme was expressed in SW1116R cells but not in HeLaK cells. Subsequently, the expression of mdr1 mRNA and Pgp decreased significantly in the transfected SW1116R cells, and was even lower than in parent non-resistant SW1116 cells. The functional ability of Pgp to facilitate rhodamine 123 (Rh123) efflux showed that the transfected SW1116R cells with low Pgp expression retained Rh123, whereas non-transfected SW1116R cells with high Pgp expression released the dye quickly. There was no difference in mdr1 mRNA or in Pgp between non-transfected and transfected HeLaK cells. Drug resistance to doxorubicin (DOX) decreased 93.1% in the transfected SW1116R cells, while no change in drug resistance occurred in the infected HeLaK cells. DOX could clearly inhibit the growth of transfected SW1116R tumors but had no effect on untransfected and on transfected HeLaK cells in vivo. These results indicate that our anti-mdr1 ribozyme is expressed only in CEA-producing colon-cancer cells and reverses their drug resistance selectively.

Co-transfection of MDR1 and MRP antisense RNAs abolishes the drug resistance in multidrug-resistant human lung cancer cells.

The resistance of lung cancer cells to the therapeutic actions of anticancer drugs is a serious clinical problem often encountered during cancer chemotherapy. It is very important, therefore, to investigate how to prevent and/or reverse this drug resistance. To this end, we took advantage of the fact that the overexpression of MDR1 and MRP genes, two genes known to be associated with the development of drug resistance, is very common in lung cancer. We used antisense RNA in an attempt to prevent expression of the protein products of these genes. Using a retrovirus, we introduced the antisense RNAs of MDR1 and MRP genes into doxorubicin-selected, multidrug-resistant GAOK cells, a cell which overexpresses both MDR1 and MRP genes. The expression levels of the products of the MDR1 gene (Pgp) and MRP gene (Mrp) in the transfected cells were analyzed using flow cytometry, and the drug resistances of the transfected cells were detected by a cell viability (MTT) assay. The expression of Pgp and Mrp in the transfected cells was almost completely inhibited by the antisense RNAs: expression levels decreased 64% and 93%, respectively. In parallel, the drug resistance of these cells decreased about 99% to doxorubicin, 98% to vinblastine, and 97% to colchicine. These results show that: a) antisense RNAs can attenuate drug resistance, an inhibition that might lead to new treatments for patients who are, or become, refractory to conventional chemotherapy; b) MDR1 and MRP appear to be cooperating to confer drug resistance in GAOK cells.

Purification of the adenomatous polyposis coli (APC) gene product.

Mutation of the adenomatous polyposis coli (APC) gene is responsible for familial adenomatous polyposis and is an etiologic factor for digestive tract malignancies. Although the APC gene product (APC) is believed to play a role in growth suppression of colonocytes, the underlying mechanism is not clear. However, recent evidence does suggest that APC is a microtubule-associated protein (MAP), and like other MAPs, it can be phosphorylated, as we have shown. To facilitate studies of APC function, we purified the APC protein. To purify the full-length APC protein, HCT116 human colon cancer cells were lysed and the particulate fraction from the lysate was extracted with ammonium sulfate followed by Sepharose 4B and DEAE-Sephacel column fractionation and then by sucrose zonal density gradient centrifugation. The final purified APC fraction was determined to be about 1000-fold enriched in APC. The availability of purified APC will be valuable in investigating possible growth-suppressing mechanisms of APC including specific sites of APC phosphorylation and APC's interaction with other cellular proteins.

Immunodetection of the presence or absence of full-length APC gene product in human colonic tissues.

In order to detect the presence or absence of wild-type adenomatous polyposis coli (APC) gene protein (APC) in human colonic tissues, we immunoaffinity purified two polyclonal rabbit antibodies (APC-1 and APC-2) directed against defined epitopes in the middle and carboxyl regions of APC. Such antibodies proved useful in western blot analysis of matched colonic mucosa and tumor sample pairs. A 300 kDa band corresponding to APC was detected in samples from normal colonic mucosa using both antibodies. No tumor samples (n = 14) showed a detectable 300 kDa band. SW480 colon carcinoma cells, known to express truncated APC lacking the carboxyl half of the protein, were also negative. These results indicate that our antibodies bind to full-length but not truncated APC. Thus, western blot analysis employing APC-1 and APC-2 antibodies may be used to evaluate the absence or presence of wild-type APC. The value of this methodology in detecting APC mutations, which mainly involve protein truncation or allelic loss, is based on its ability to demonstrate negative or reduced level of immunoreactivity toward full-length APC in tissues that contain such mutations.

Phosphorylation of the adenomatous polyposis coli protein and its possible regulatory effects in cells.

The adenomatous polyposis coli (APC) gene is etiologically associated with familial adenomatous polyposis and gastrointestinal malignancies, but its cellular function and role in tumorigenesis are unclear. Recent reports indicate that wild-type, but not mutant, APC gene product (APC) is associated with and promotes the assembly of cytoskeletal microtubules in vitro, suggesting that this mechanism has importance in tumor development. Because other microtubule-associated proteins (MAPs) undergo phosphorylation in their normal functioning, we postulated that APC is a phosphoprotein. HCT116 cells, containing full-length APC protein, were [32P]-prelabeled, and a 300-kDa band corresponding to phosphorylated APC was immunoprecipitated using each of three different anti-APC antibodies. High voltage electrophoresis of [32P]-labeled APC showed the presence of phospho-serine and phospho-threonine residues. Further immunoprecipitation analyses showed phosphorylation of i) full-length APC in human lymphoblastoid cells and ii) carboxyl-truncated APC in SW480 and DiFi colon carcinoma cells. Thus, APC is probably a phosphoprotein in normal and malignant tissues. We hypothesize a mechanism whereby phosphorylation of APC may play a regulatory role in its interaction with microtubules. This may involve phosphorylation of (Ser/Thr)-Pro amino acid motifs in APC's basic domain. We propose that deletion of this domain disrupts APC binding to microtubules, explaining how APC mutations are linked to cancer development.

Radioimmunoassay of the APC gene product using antibodies against its middle and carboxyl regions.

A radioimmunoassay (RIA) has been developed for the adenomatous polyposis coli protein (APC). High-avidity rabbit polyclonal antibodies were produced against synthetic peptides corresponding to amino acids 1865-1881 (APC-1) and to amino acids 1336-1350 (APC-2) in APC's 2844 amino acid sequence. Both antibodies were utilized in RIA to evaluate full-length APC that is present in the insoluble particulate fraction of cell lysates. High salt extraction, often employed for coiled-coil type protein preparations, was found to be useful for extraction of APC from lysates of normal colonic epithelium. Proteolytic digestion of high salt extracts increased antibody reactivity toward both epitopes, suggesting that APC-1 and APC-2 antigenic sites are partially concealed due to APC's involvement in multiprotein complexes. Thus, RIA using our antibodies will provide a valuable tool for APC protein purification and in studies for elucidating APC's biologic function.

Positron emission tomography for preoperative staging of colorectal carcinoma.

PURPOSE: Positron emission tomography (PET) is an imaging technique based on in vivo cellular metabolism. Increased glucose metabolism in neoplastic cells is detected by using fluorine-18 deoxyglucose. In an ongoing pilot study to determine the usefulness of this technique, PET is compared with computerized tomography (CT) for the preoperative staging of colorectal carcinoma. METHODS: Sixteen patients were evaluated with both PET and CT of the abdomen and pelvis. Results were compared with operative and histopathologic findings. Fifteen malignant lesions were found in 16 patients by histology. PET had a positive predictive value of 93 percent and a negative predictive value of 50 percent. By comparison CT had a positive predictive value of 100 percent and a negative predictive value of 27 percent. CONCLUSIONS: These preliminary results indicate that PET has increased sensitivity for staging colorectal carcinoma, whereas CT has higher specificity. The predictive value of a positive PET compares favorably with CT. Furthermore, the predictive accuracy for detection of colorectal carcinoma is 83 percent for PET and 56 percent for CT.

Biologic functions of the genes responsible for familial cancer.

Clinical and molecular genetic studies on human familial cancers have provided important information toward further understanding of the etiology of hereditary and sporadic cancer. Our next challenge is to determine how mutations involving this group of genes leads to functional changes in the cell causing malignant growth. This article summarizes some of the significant findings recently reported concerning the biologic function of the genes that are involved in hereditary cancer syndromes.

Down-modulation of epidermal growth factor receptor accompanies TNF-induced differentiation of the DiFi human adenocarcinoma cell line toward a goblet-like phenotype.

Although the biologic response modifier tumor necrosis factor-alpha (TNF) is a known differentiation inducer in hematopoietic cells, its role in differentiation of other tissue types has yet to be elucidated. In the studies presented here, TNF treatment of the human rectal adenocarcinoma cell line, DiFi, elicits characteristics of early stage differentiating, mucin-producing colonocytes. Not only are TNF-treated DiFi cells growth-inhibited by TNF, but they also display a unique morphology. Additionally, TNF treatment of DiFi cells enhances > fivefold the expression of high molecular weight mucin glycoproteins, as measured by [125I]-wheat germ agglutinin (WGA) binding and the human milk fat globule-1 (HMFG-1) anti-MUC1 antibody reactivity. The induction of these differentiation characteristics correlates with novel alterations in epidermal growth factor receptor (EGF-R). Following 5-day TNF treatment of DiFi cultures, EGF receptor levels, kinase autophosphorylation activity, and receptor tyrosine phosphorylation are reduced by > fourfold. The establishment of a model system in which goblet-like cell characteristics and alterations in a growth factor receptor can be induced in vitro may be potentially useful in studying the underlying mechanisms of colonic epithelial cell proliferation and differentiation.

Retinoblastoma gene product-associated proteins in human colon cancer cell lines.

The expression of cellular proteins that associate with the human retinoblastoma (RB1) gene protein was examined in three colorectal carcinoma cell lines (SW480, SW403, DiFi) by immunoprecipitation analysis, using the mouse monoclonal antibody (RB-MAb-1) directed against the RB1 gene product (RB). The potential of RB-MAb-1 to detect RB-associated proteins by immunoprecipitation analysis was confirmed using three control cell lines (WI-38, J82, T24) known to express RB and/or RB-associated proteins. In all colon cancer cell lines tested, multiple RB-associated protein (RAP) bands were found with molecular weights of 30-50 kDa. Because involvement of the RB1 gene in colorectal tumorigenesis appears to differ from its role in other cancer types (wherein RB1 is lost or inactivated), it will be important to characterize the role of RAPs in RB growth regulatory mechanisms of colonic epithelial cells.

Synthetic retrotransposon vectors for gene therapy.

New gene therapy methods are rapidly being developed to permit the expression of tumor suppressor genes, cytotoxins, anticancer antigens, and immunoregulatory proteins in the treatment of cancer. Large-scale testing in humans has been delayed by questions concerning the safety and effectiveness of preferred retroviral vectors and helper cells. These vector systems are limited by their ability to undergo homologous recombination with endogenous retroviruses or helper-viral sequences, resulting in release of replication-competent retrovirus (RCR). In addition, transcriptional inactivation of the retroviral promoter often occurs, caused in part by methylation of CpG islands in the retroviral long terminal repeats (LTRs). We report the production of highly specific retrovectors using gene amplification together with oligonucleotide building blocks. The synthetic vectors were based on mouse VL30 retrotransposon NVL3, and lacked homology to retroviral helper gene sequences. Three of four constructs made by gene amplification yielded biologically active vectors. These constructs efficiently transmitted and stably inserted a neomycin resistance marker gene into the genome of recipient cells, expressing an abundant RNA species of the expected size in the absence of detectable replication competent retrovirus. The vectors and techniques described enable widely applicable expression modes using generic helper cells, and require only approximately 1.3 kb of cis-acting vector RNA sequences for faithful transfer and expression of genetic material.

RB1 protein in normal and malignant human colorectal tissue and colon cancer cell lines.

The pattern of human retinoblastoma (RB1) gene protein expression was directly examined in normal and malignant human colorectal tissues and in seven colorectal carcinoma cell lines by immunohistochemistry using the mouse monoclonal antibody (RB-MAb-1) directed against the retinoblastoma protein (RB). This is the first demonstration of RB immunostaining in adult human colonic epithelium and colorectal carcinomas. Specificity using RB-MAb-1 was confirmed by western blot analysis, which showed bands of 110-116 kDa corresponding to the sizes of unphosphorylated and phosphorylated RB. RB staining of normal adult colonic epithelium was confined to the nucleus and was most intense in the transitional zone of the crypt, whereas lumenal cells (fully differentiated) were RB negative. Primary colorectal carcinomas and all the colon cancer cell lines stained positively for nuclear RB, but the expression was heterogeneous with varying fractions of RB negative cells present. Because we and others have previously shown that loss or inactivation of the RB1 gene is infrequent in colorectal carcinomas, reduced RB expression in such cells is probably due to a cellular regulatory mechanism. For example, RB negative cells may be those in early-G1 phase (known to have reduced RB levels) or growth-arrested cells that have differentiated. The ability to directly detect RB in primary colorectal carcinomas will permit assessment of whether heterogeneous expression of the RB1 gene product has prognostic significance for survival of patients with this cancer.