Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions.

OBJECTIVE: The objective of this study was to establish a flow cytometry assay for measuring c-FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. METHODS: Two c-FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c-FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal-to-noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c-FLIP expression was analysed for association with clinicopathological parameters and survival. RESULTS: Rabbit polyclonal c-FLIP by Abcam and the IntraStain kit by Dako performed best. c-FLIP expression was detected in tumour cells in all 69 effusions (expression range 21-100%, median = 80%). No association was found between c-FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c-FLIP levels and expression of the previously studied apoptosis marker cleaved caspase-3 (P = 0.029). CONCLUSIONS: An assay for measuring c-FLIP in cytology specimens is presented. c-FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.

COX-2 expression in striated muscle under physiological conditions.

OBJECTIVES: The role of cyclooxygenase-2 (COX-2) in disease has been extensively studied, (Annu Rev Med (2002) 53:35; N Engl J Med (2001) 345:433) but less information is available with respect to possible physiological functions of COX-2. Information on how and where COX-2 is expressed under physiological conditions may increase our understanding of its physiological role. Previous studies have revealed a COX-2 dependent production of prostanoids under physiological conditions, without entirely determining the source of this production. MATERIALS AND METHODS: To assess COX-2 expression under normal conditions, we analyzed tissue specimens that were removed from 30 healthy study subjects in conjunction with surgical procedure related to insertion of dental implants and from three patients which had muscle tissue from Quadriceps femoris muscle removed as part of surgical treatment of soft tissue sarcomas not directly affecting the muscle tissue. Immunohistochemistry and immunoblotting (Western blotting) was used to assess the presence of COX-2 protein. RESULTS: In 25 of 30 patients (83%), COX-2 protein was expressed in striated muscle, as assessed by immunohistochemistry. All cases had COX-2 expression verified by Western blotting. In none of the 25 subjects with COX-2 expression did we notice concomitant inflammation of the adjacent submucosal tissue. CONCLUSIONS: It is a novel finding that COX-2 is expressed in striated muscle under physiological conditions. COX-2 activity in striated muscle is a possible explanation for the hitherto unknown localization of prostanoids synthesis under physiological conditions.

Cyclin A expression in superficial spreading malignant melanomas correlates with clinical outcome.

The present study analysed by immunohistochemistry the protein level of cyclin A and Ki-67 in a panel of paraffin-embedded tissue obtained from 172 primary (110 superficial and 62 nodular) and 73 metastatic melanomas, and ten benign naevi. Since cyclin A exists in the same quaternary complex in the S-phase of the cell cycle as the cdk inhibitor p21WAF1/CIP1, the levels of the two proteins were compared. Cyclin A and Ki-67 were heterogeneously expressed in the malignant tumours, whereas in benign naevi, only rare positive cells were detected. In superficial spreading melanomas, the cyclin A level was related to tumour thickness, with less expression in thinner lesions (p<0.00001), and to Ki-67 (p<0.00001) and p21WAF1/CIP1 (p=0.01) scores. Multivariate analysis showed that in addition to the depth of the primary tumour, the protein level of cyclin A was an independent indicator of relapse-free period (thickness, p<0.00001; cyclin A, p=0.0003). In contrast, in nodular melanoma, the cyclin A level was associated with Ki-67 expression, but neither cyclin A nor Ki-67 was related to tumour thickness (cyclin A, p=0.06; Ki-67, p=0.61) and neither had any impact on relapse-free (cyclin A, p=0.64; Ki-67, p=0.32) or overall (cyclinA, p=0.94; Ki-67, p=0.45) survival. In conclusion, the results indicate that cyclin A is a strong prognostic factor for patients with superficial spreading melanomas. In nodular melanomas, the proliferation rate seems to have little impact on disease progression.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

Expression of cell cycle proteins in ovarian carcinoma cells in serous effusions-biological and prognostic implications.

OBJECTIVE: The aim of this study was to investigate the expression of cell cycle proteins in ovarian carcinoma cells in serous effusions and respective solid tumors. METHODS: Fifty-five malignant effusions and 38 tumors (20 primary, 18 metastatic) were immunohistochemically stained for cyclin A, p27(kip1), and Ki-67. Staining extent (0-100% cells) and intensity (0-3 scale) were scored. Cyclin A and p27(kip1) expression was additionally studied in 29 malignant effusions using immunoblotting. Immunohistochemistry results in effusions were evaluated for possible association with clinicopathologic parameters. RESULTS: Nuclear immunoreactivity for all markers was detected on carcinoma cells in the majority of effusions using immunohistochemistry. Similarly, immunoblotting showed the presence of cyclin A and p27(kip1) in 29/29 and 25/29 specimens, respectively. Intense (3) immunoreactivity for Ki-67 was detected more often in peritoneal effusions, compared with those of pleural location (P = 0.036). Staining in primary and metastatic lesions was generally comparable to that of tumor cells in effusions. Staining for p27(kip1) was more diffuse in effusion specimens obtained prior to the institution of chemotherapy (P = 0.042). In an analysis of all effusions, an association was observed between the number of cells that were immunoreactive for Ki-67, cyclin A, and p27(kip1) (cyclin A-Ki-67: P = 0.008; p27(kip1)-Ki-67: P = 0.019; cyclin A-p27(kip1): P = 0.032). In survival analysis, the presence of more diffuse (P = 0.042) and intense (P = 0.019) staining for cyclin A correlated with prolonged overall survival. CONCLUSIONS: The expression of the studied cell cycle markers does not differ markedly between ovarian carcinoma cells in the pleural and peritoneal cavity, supporting our previous studies of several metastasis-associated molecules. The presence of cyclin-A-positive cell populations is associated with a more favorable disease outcome, possibly due to the targeting of proliferating cells by chemotherapeutic agents. However, the decline in the fraction of p27(kip1)-positive cells in posttreatment specimens may point to additional mechanisms involved in this selection.

Reduced expression of p21WAF1 is an indicator of malignant behaviour in anal carcinomas.

AIMS: p21 and p27 protein expression were examined in a comparatively large series of patients with squamous cell carcinoma of the anal canal and compared with clinical and histopathological data (tumour stage, nodal status and differentiation). METHODS AND RESULTS: We analysed the expression of p21 and p27 protein in 94 anal carcinomas by immunohistochemistry. Nuclear p21 and p27 staining were detected in 71% (67/94) and 75% (71/94) of the cases, respectively. There was no significant association between p27 staining and tumour stage, nodal status or overall survival. We observed that negative p21 immunoreactivity was significantly associated with poorly differentiated anal carcinomas. Furthermore, a shorter overall survival for patients with no p21 protein expression was seen. CONCLUSIONS: Our data indicate that p21 levels, but not p27 expression, may be a useful predictor of survival in patients with anal carcinomas.

Levels of cyclin D1 and D3 in malignant melanoma: deregulated cyclin D3 expression is associated with poor clinical outcome in superficial melanoma.

We examined 172 primary (110 superficial and 62 nodular) and 73 metastatic melanomas, as well as 10 benign nevi, for protein expression of cyclin D1 and cyclin D3 and evaluated the relationship between deregulated protein levels and clinical outcome. For both proteins, a heterogeneous nuclear staining pattern was observed. Cyclin D3 was expressed by 96% of primary and 97% of metastatic melanomas. The corresponding percentages for cyclin D1 were 62% and 29%, respectively. In benign nevi, only rare cyclin D3-positive cells and no cyclin D1-positive cells were observed. High levels of cyclin D3 (>5% of the cells stained) were detected in 26 of 62 (42%) nodular melanomas and in 22 of 110 (20%) superficial tumors, whereas no such difference was observed with respect to cyclin D1. In superficial melanomas, a significant concordant staining pattern was observed between cyclin D1 and cyclin D3 (P = 0.0009), cyclin D1 and Ki-67 (P = 0.0001), cyclin D1 and cyclin A (P = 0.02), cyclin D3 and Ki-67 (P < 0.00001), and cyclin D3 and cyclin A (P = 0.002). Kaplan-Meier analysis revealed that high levels of cyclin D3 were an indicator of early relapse and decreased overall survival for patients with superficial (P = 0.001 and P = 0.009, respectively) but not nodular (P = 0.64 and P = 0.23) melanoma. Cyclin D1 did not have any impact on disease-free and overall survival for either of the subtypes. In conclusion, our results suggest that deregulation of cyclin D3 expression leading to increased proliferation may be a prognostic factor for superficial melanoma, whereas deregulated cell cycle machinery seems to have little impact on disease progression of nodular melanoma.

Up-regulation of ephrin-A1 during melanoma progression.

Ephrin-A1, formerly called B61, is a new melanoma growth factor; it is angiogenic and chemoattractant for endothelial cells. EPH-A2, or ECK (a receptor for ephrin-A1), is ectopically expressed in most melanoma cell lines; the pathology where this expression is first manifested and the possible role of the receptor in tumor progression are unknown. To determine these, we studied the expression of this ligand and receptor in biopsies of benign and malignant melanocytic lesions. EPH-A2 was not detected in normal melanocytes, benign compound nevi or advanced melanomas, though it was found in 2 of 9 biopsies of malignant melanoma in situ. Ephrin-A1 was present in occasional early lesions and in advanced primary melanomas (43%) and metastatic melanomas (67%). Expression of ephrin-A1 was induced in melanoma cells by pro-inflammatory cytokines. Our findings are consistent with 2 possible roles for ephrin-A1 in melanoma development: it may promote melanocytic cell growth or survival and induce vascularization in advanced melanomas. Both effects may be potentiated by inflammatory responses. Our data are consistent with earlier observations that an inflammatory infiltrate is associated with poor prognosis in thin primary melanomas.

Interleukin-6 dependent induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 is lost during progression of human malignant melanoma.

Human melanoma cell lines derived from early stage primary tumors are particularly sensitive to growth arrest induced by interleukin-6 (IL-6). This response is lost in cell lines derived from advanced lesions, a phenomenon which may contribute to tumor aggressiveness. We sought to determine whether resistance to growth inhibition by IL-6 can be explained by oncogenic alterations in cell cycle regulators or relevant components of intracellular signaling. Our results show that IL-6 treatment of early stage melanoma cell lines caused G1 arrest, which could not be explained by changes in levels of G1 cyclins (D1, E), cdks (cdk4, cdk2) or by loss of cyclin/cdk complex formation. Instead, IL-6 caused a marked induction of the cdk inhibitor p21WAF1/CIP1 in three different IL-6 sensitive cell lines, two of which also showed a marked accumulation of the cdk inhibitor p27Kip1. In contrast, IL-6 failed to induce p21WAF1/CIP1 transcript and did not increase p21WAF1/CIP1 or p27kip1 proteins in any of the resistant lines. In fact, of five IL-6 resistant cell lines, only two expressed detectable levels of p21WAF1/CIP1 mRNA and protein, while in three other lines, p21WAF1/CIP1 was undetectable. IL-6 dependent upregulation of p21WAF1/CIP1 was associated with binding of both STAT3 and STAT1 to the p21WAF1/CIP1 promoter. Surprisingly, however, IL-6 stimulated STAT binding to this promoter in both sensitive and resistant cell lines (with one exception), suggesting that gross deregulation of this event is not the unifying cause of the defect in p21WAF1/CIP1 induction in IL-6 resistant cells. In somatic cell hybrids of IL-6 sensitive and resistant cell lines, the resistant phenotype was dominant and IL-6 failed to induce p21WAF1/CIP1. Thus, our results suggest that in early stage human melanoma cells, IL-6 induced growth inhibition involves induction of p21WAF1/CIP1 which is lost in the course of tumor progression presumably as a result of a dominant oncogenic event.

Collateral expression of proangiogenic and tumorigenic properties in intestinal epithelial cell variants selected for resistance to anoikis.

Although in vitro anchorage-independent growth is widely used as a marker of cell transformation, the biological implications of this trait are poorly understood. We previously demonstrated that enforced anchorage-independent growth of a nontumorigenic, immortalized epithelial cell line (IEC-18) in multicellular spheroid culture results in massive apoptotic cell death. This death process, termed anoikis, is prevented by expression of transforming oncogenes, which also confer tumorigenic competence. This study examines whether acquisition of an anoikis-resistant phenotype is causally related to the tumorigenic capacity of transformed epithelial cells. Parental IEC-18 cells were subjected to 10 cycles of selection for survival in speroid culture. Unlike parental cells, the resulting anoikis-resistant variants (AR1.10 and AR2.10) formed relatively large tumors in nude mice. Both anoikis-resistant sublines displayed upregulated expression of vascular endothelial growth factor (VEGF), a potent angiogenesis stimulator. VEGF121 overexpression alone did not induce tumorigenic conversion of parental IEC-18 cells, which remained highly susceptible to anoikis. We postulate that both anoikis-resistance and angiogenic-competence contribute to tumor formation. Development of anoikis-resistance can be then viewed as a precondition for expression of the tumorigenic phenotype. Our results suggest that even when angiogenesis is not a rate limiting factor (e.g. in vitro) the selective pressures of solid tumor-like, 3-dimensional growth conditions favoring anoikis resistance result in collateral induction of a proangiogenic phenotype.

E-Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27(KIP1).

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell- cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, we engineered E-cadherin-positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.

Protein expression of the cell-cycle inhibitor p27Kip1 in malignant melanoma: inverse correlation with disease-free survival.

In the present study we analyzed, by immunohistochemistry, a panel of human melanomas for protein expression of the cyclin-dependent kinase (cdk) inhibitor p27Kip1 and evaluated whether deregulated expression correlates with clinical outcome for this type of cancer. We found that p27Kip1 was strongly expressed by normal melanocytes and benign nevi, whereas in malignant melanoma, a heterogeneous expression pattern was observed. In the case of nodular melanomas, the level of p27Kip1 was found to correlate significantly with the thickness of the tumor, with less protein expressed in thicker lesions. We also found that patients having tumors with fewer than 5% p27Kip1-staining cells had a significantly higher risk of early relapse of their disease compared with those expressing moderate or high levels. In contrast, the level of p27Kip1 did not correlate with tumor thickness or disease-free survival in patients with superficial spreading melanomas, suggesting that p27Kip1 may play different roles in these two major pathological subgroups of malignant melanoma. Furthermore, p27Kip1 did not appear to have an influence on overall survival for either subgroup. When we examined the combined effect of p21WAF1/CIP1 (another cdk inhibitor) and p27Kip1 on clinical outcome, we found that analysis of these two cdk inhibitors together may have greater prognostic potential than either alone. In conclusion, our results suggest that virtually complete loss of p27Kip1 protein expression has potential importance as a prognostic indicator of early relapse in patients with nodular melanoma The results, furthermore, underscore the value of analyzing multiple cell cycle regulatory proteins to obtain the most reliable indication of prognosis.

Differential expression patterns of S100A2, S100A4 and S100A6 during progression of human malignant melanoma.

Three members of the S100 gene family, S100A2, S100A4 and S100A6, have been suggested to be associated with cancer development and metastasis. To study their involvement in the tumorigenesis of human melanoma, we examined the mRNA expression levels of the 3 genes in 45 melanoma metastases and in 20 benign nevi. Interestingly, whereas none of the metastases expressed S100A2 mRNA, and the expression level was low in 6 cell lines established from primary melanomas, all nevi showed moderate to high expression levels. Our results suggest that loss of S100A2 gene expression may be an early event in melanoma development. A significant correlation was found between the expression of S100A6 in melanoma metastases and both the survival time of the patients and the thickness of the corresponding primary tumors. For the S100A4 gene, however, no relationship was found between gene expression and clinical parameters of melanoma malignancy. The observed differences in expression patterns of the 3 S100 genes suggest distinct roles of their products in melanoma tumorigenesis and/or metastasis, and the results encourage studies to evaluate the potential value of using S100A2 and S100A6 expression levels as markers in the clinical management of melanoma.

Loss of expression of receptor tyrosine kinase family genes PTK7 and SEK in metastatic melanoma.

Protein tyrosine kinases (PTKs) have been implicated in the development of many common human tumours including melanoma. Previously we isolated PTK gene sequences expressed in normal melanocytes. Here we examined expression of 9 of these genes in cell lines derived from defined stages of melanoma progression, by Northern blotting and in some cases immunoblotting. We also tested cells from 2 animal models of particular stages in progression, as well as uncultured biopsies of metastatic melanoma. The expression of 2 receptor kinase family members found in melanocytes, PTK7/CCK-4 and SEK/TYRO1, was decreased or lost in advanced melanomas. PTK7 mRNA was found in only 54% of melanoma cell lines and 20% of melanoma biopsies. Similarly, expression was lost in 2 advanced cell lines selected from an early melanoma line that did express PTK7 mRNA. SEK/TYRO1 expression was observed in 75% and 17% of cell lines from primary and metastastic melanomas, respectively. Conversely, mRNA for the non-receptor kinase PTK6/BRK was not detected in normal melanocytes or primary melanoma lines, but was found in 9% of metastatic melanoma cell lines.

High levels of messenger RNAs for tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in primary breast carcinomas are associated with development of distant metastases.

Tissue inhibitors of metalloproteinases (TIMPs) are believed to possess several cellular functions, particularly the contrasting activities of inhibiting tissue-degrading enzymes and promoting cellular growth. In attempts to elucidate which of these functions may prevail in breast cancer, expression of mRNAs for TIMP-1 and TIMP-2 in the primary carcinomas from 34 breast cancer patients was related to known prognostic parameters and the clinical outcome. High levels of TIMP-1 mRNA showed significant correlation with the presence of lymph node metastases (P = 0.0067), development of distant metastases (P = 0.014), and early death of the disease (P = 0.020). Elevated expression of TIMP-2 mRNA was associated with development of distant metastases (P = 0.0055). No correlations, however, were observed between mRNA levels of TIMPs and prognostic factors such as patient age, tumor size, grade of anaplasia, or steroid receptor status; neither were any correlations found between these clinicopathological characteristics and the mRNA expression of the collagenolytic enzymes matrix metalloproteinase-2 and matrix metalloproteinase-9. The present data suggest that high levels of TIMP-1 and TIMP-2 mRNAs in the primary carcinomas are strongly associated with development of metastasis in breast cancer.

Expression of stromelysin-3 in medullary carcinoma of the breast.

Matrix metalloproteinases are belived to play a central role in the invasion and metastasis of human cancers by mediating the degration of extracellular matrix components. Expression of stromelysin-3 (ST3), a new member of matrix metalloproteinase family, was investigated by in situ hybridization in 32 tissue samples of medullary carcinoma of the breast. Eighty-four per cent of the cases showed ST3 gene expression in stromal cells adjacent to tumor cells. Therefore, expression of ST3 in stromal cells may be expected to play a significant role in the destruction of extracellular matrix and invasion of medullary carcinoma of the breast.

Cyclin kinase inhibitor p21WAF1/CIP1 in malignant melanoma: reduced expression in metastatic lesions.

Immunohistochemical analysis of the expression of the cyclin kinase inhibitor p21WAF1/CIP1 in a panel of primary and metastatic human melanocytic tumors was performed. It was found that, independent of the p53 status, approximately 30% of the primary melanomas and 40% of the metastases completely lacked expression of this cell cycle inhibitor. Some tumors were also analyzed by Northern blotting, and in most of the cases a consistant correlation between mRNA and protein expression was observed. In four benign nevi studied, WAF1/CIP1 mRNA was expressed whereas the protein was not detected, suggesting a post-transcriptional regulation of the inhibitor in these cases. In superficial spreading melanomas, a significant correlation between protein expression and tumor thickness was found, with thin lesions showing low protein levels. Interestingly, by comparing primary and metastatic specimens obtained from the same patient, a reduction in p21WAF1/CIP1 antibody staining was observed in the latter, probably reflecting a more aggressive phenotype of the metastases. In conclusion, our results demonstrate the complexity in the relationship between p21WAF1/CIP1 expression and tumor phenotype and furthermore suggest that aberrant expression of the cyclin-dependent kinase inhibitor may be of importance in the development and progression of sporadic malignant melanoma.

TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1.

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.

Reversal of the in vivo metastatic phenotype of human tumor cells by an anti-CAPL (mts1) ribozyme.

The putative role of the CAPL gene in enhancing the development of human cancer metastasis was examined by transfecting human high-expressing osteosarcoma cells with a hammerhead ribozyme directed against the gene transcript. The ability of the ribozyme to cleave target mRNA in intact cells was demonstrated in a 5'-rapid amplification of cDNA ends assay. In transfected cells, a suppression of the capacity to give skeletal metastases upon intracardial injection into nude rats was observed in cell clones with reduced expression of CAPL mRNA and protein, whereas in vitro and in vivo cell proliferation and tumorigenicity were unchanged. The results provide direct evidence that the expression level of the CAPL-encoded protein can determine the metastatic potential of osteosarcoma cells, and they demonstrate an association between reduced gene expression and proliferation-independent inhibition of the metastatic capacity of human tumor cells. The effects of the specific cleavage of CAPL mRNA indicate that the gene product is involved in key cellular functions associated with the metastatic process and suggest that therapeutic modulation of the protein function may represent a novel approach for inhibiting the metastatic spread of cancer cells.

Induction and reversal of cell adhesion-dependent multicellular drug resistance in solid breast tumors.

Although there are a number of chemotherapeutic drugs available for the treatment of breast cancer, eg. adriamycin, cyclophosphamide and taxol, their effectiveness is severely limited by expression of intrinsic resistance in some patients and by acquired resistance in others. There is thus an urgent need to develop innovative methods to try and make these drugs more effective than is currently the case. One such method is to combine them with novel "chemosensitizers", i.e., drugs which themselves lack anti-tumor cytotoxic properties but which will increase the efficacy of those which do. In this regard we hae been studying the hypothesis that the resistance of solid tumors, including breast cancer, can be expressed at the prototissue/multicellular level, and that this "multicellular resistance" can be minimized or reversed by the appropriate use of so-called "anti-adhesive" agents. RESULTS/BACKGROUND: It is well known that monolayer cultures of tumor cells-including murine breast cancer-are generally much more intrinsically chemosensitive than the same cells grown as solid tumors in vivo. However, the relative resistance of solid tumors can often be recapitulated in tissue culture simply by growth of the tumor cells as three dimensional multicellular spheroids. There are cases where this is also true with respect to acquired drug resistance. This "multicellular resistance" could be due to such factors as insufficient drug penetration, a reduced growth fraction, or a decreased sensitivity to drug induced apoptosis mediated by cell-cell interaction survival signals. Can such multicellular resistance mechanisms in solid tumors be reversed? With respect to this question, we have recently found that the relative intrinsic resistance of intact murine EMT-6 mouse mammary carcinoma spheroids can be significantly reversed by the anti-adhesive (disaggregating) effects of hyaluronidase. Moreover, this novel method of chemosensitization appears to depend on increased recruitment of disaggregated cells into the cycling pool, thus rendering them more sensitive to a cell cycle dependent drug such as cyclophosphamide. The reduced growth fraction observed in spheroids appears to be due to a marked cell contact-dependent upregulation of the cyclin dependent kinase inhibitor, p27Kipl. FUTURE OBJECTIVE: The overall goal of our current and future research is to determine whether solid tumors, including human breast cancer, express intrinsic or acquired resistance at the multicellular level to such drugs as taxol or cyclophosphamide, and if so, determine whether it can be reversed by the chemosensitizing effect of anti-adhesive agents. This will require a search for effective anti-adhesive agents for human cancers as hyaluronidase has not been found to possess anti-adhesive function against such tumors to date. In addition, the counter-intuitive and innovative idea of downregulating p27kipl in human breast cancers as a means of cytotoxic drug chemosensitization is also being evaluated.