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Background Basal cell carcinoma (BCC) is the most common form of skin carcinoma. Aim To investigate the function of key micro(mi)RNAs and to explore the potential molecular mechanisms involved in BCC. Methods The microarray dataset GSE34535, which comprises seven BCC samples and seven control samples, was downloaded from the Gene Expression Omnibus database. Differentially expressed miRNAs (DE-miRNAs) were identified. We collected tissue samples from 20 patients with BCC and 20 healthy controls (HCs), to compare the miR-18a expression in their tissue samples. Expression of miR-18a in A431 and HaCaT cells was also assayed. Following this, we upregulated and downregulated miR-18a expression in A431 cells to examine the effects on cell proliferation, migration and apoptosis. To further investigate the relative mechanism, the proteins LC3, Beclin 1, Akt and mammalian target of rapamycin (mTOR) were examined by quantitative real-time PCR and Western blotting. For further verification, we examined the expression of LC3 in the 20 BCC and 20 HC tissue samples. Results In total, 19 DE-miRNAs (13 upregulated and 6 downregulated) that were common to the BCC and HC groups were identified. Levels of miR-18a were about three-fold higher in BCC tissues and A431 cells compared with their respective control groups. In vitro, downregulation of miR-18a was shown to inhibit cell proliferation and activate autophagy via the Akt/mTOR signalling pathway, while upregulation of miR-18a promoted proliferation of these cells. LC3 was decreased in BCC compared with HC tissue samples. Conclusions Our data support an oncogenic role of miR-18a through a novel Akt/mTOR/Beclin 1/LC3 axis, and suggest that the antitumour effects of miR-18a inhibitor may make it suitable for BCC therapy.

Background: The dysregulation of epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of various malignancies including glioblastoma and some epithelial cancers. Oncogenesis occurs from the overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287–302 loop in the extracellular domain is exposed completely on EGFR variant III (EGFRvIII), partially exposed on some cancers but cryptic on normal cells. We report on the generation of antibodies to this loop.Methods: The 286–303 peptide was coupled chemically to keyhole limpet hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V regions were cloned and sequenced. Receptor binding was characterized using enzyme-linked immunosorbent assay and flow cytometry. A recombinant immunotoxin was generated from the 40H3 antibody and its cytotoxic activity characterized on relevant cancer cell lines.Results: Seven monoclonal antibodies were generated to the 287–302 loop and characterized further. Each one reacted with EGFRvIII but not wild-type EGFR. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-MB-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be cytotoxic to MDA-MB-468, A431 and F98npEGFRvIII expressing cells.Conclusions: Immunization with a peptide corresponding to a cryptic epitope from EGFR can produce tumor cell-binding antibodies. The 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be further developed as a therapeutic agent.

Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities.

UNLABELLED Although monoclonal antibodies have been used not only as analytical tools but also as biologic therapeutics, they cannot target intracellular proteins due to their large molecular size and low membrane permeability, which limit their applications. During previous attempts to delivery antibodies intracellularly, the low efficiency of escape from endosomes to the cytosol reduced the bioavailability of antibodies or antibody-conjugated effectors. Recently, we found that the fusogenic peptides (FPs) B18 and B55 from bindin, a sea urchin gamete recognition protein, facilitated the endosomal escape of FP-fused enhanced green fluorescent protein (eGFP) and/or of co-administered cargos such as dextrans [Niikura et al. A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape. J. CONTROL Release 2015;212:85-93]. In this study, we constructed FP-fused anti-epidermal growth factor receptor (EGFR) single-chain Fv (αEGFR[scFv]) proteins and evaluated their endosomal escape efficiency by utilizing a nuclear localization signal). When the FP-fused αEGFR[scFv] proteins were incubated with A431 cells, the estimated endosomal escape efficiency of αEGFR[scFv]-B18 was significantly higher than that of αEGFR[scFv] alone, suggesting that the B18 peptide facilitates endosomal escape of the conjugated scFv in cis. Moreover, αEGFR[scFv]-B55 promoted the intracellular uptake of co-administered eGFP and dextrans in trans. These results imply that B18- and B55-fused antibodies may be useful for the cell-specific intracellular delivery of biomacromolecules.

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics.

EHD3 [Eps15 homology (EH) domain-containing protein 3] is a protein that resides in tubular and vesicular membrane structures and participates in endocytic recycling, although all its functions are unknown. Since Ehd3 is most abundantly expressed in brain tissues, we examined its role in brain cancer progression. Using immunohistochemistry, we report loss of EHD3 expression in gliomas, including low-grade astrocytomas, suggesting that this is an early event in gliomagenesis. EHD3 expression is also very low in most of glioma cell lines tested. In two cell lines, a bisulfite sequencing method identifies promoter hypermethylation as a mechanism of Ehd3 silencing, and its expression was restored by the demethylating agent 5-Azacytidine. Doxycycline-inducible restoration of EHD3 expression to glioma cells decreases their growth and invasiveness and induces cell cycle arrest and apoptosis. Furthermore, shRNA-mediated Ehd3 silencing increases cell growth. Using a xenograft model, we demonstrate Ehd3 growth inhibitory functions in glioma cells in vivo. We suggest that Ehd3 functions as a tumor suppressor gene and loss of its expression is a very common event in gliomas. This is the first study to highlight the importance of a member of the C-terminal EHD proteins in cancer and to link their functions to the cell cycle and apoptosis.

Kruppel-like factor 4 (KLF4) is a transcription factor that is highly expressed in differentiated epithelial cells including that of the skin. It is critical for specification or function of differentiated epithelial cells. Moreover, KLF4 functions either as a tumor suppressor or an oncogene depending on different cellular contexts. However, the role of KLF4 in skin tumorigenesis remains controversial. To address this issue, we first examined KLF4 expression using a cohort of samples from patients with skin squamous cell carcinoma and basal cell carcinoma and found that in 21 of 24 tumor tissues (87.5%), KLF4 expression as assayed by immunohistochemistry was absent when compared with that in normal tissues. In addition, knockdown of KLF4 in human epidermal squamous cell carcinoma SCC13 cells was accompanied by increased cell growth. Further analysis revealed that KLF4 deficiency promoted cell migration and adhesion, which are the important properties of tumor cells. These observations were supported by the effect upon overexpression of KLF4 in SCC13 cells. Furthermore, we generated a novel tamoxifen-inducible KLF4/CreER and KLF4(flox) double transgenic mouse model to examine the role of KLF4 in skin cancer development. Consistent with in vitro studies, KLF4 deficiency increased the ability of migration and adhesion of mouse primary skin keratinocytes. Moreover, KLF4 knockout led to increased cell proliferation and skin carcinogenesis in a classical DMBA/TPA mouse skin cancer model. Taken together, our data suggest that KLF4 inhibits cell proliferation, migration and adhesion and that loss of KLF4 promotes skin tumorigenesis.

The Ras proto-oncogene mediates a wide variety of cellular events and is frequently mutated in cancer. MicroRNAs (miRNAs) may regulate the development of cancer through their effect on the target genes. In the search of miRNAs that target on Ras, miR-18a* is the first time confirmed to target on K-Ras and furthermore not on N- and H-Ras. miR-18a* repression by transfection with anti-miR-18a* inhibitor increased the K-Ras expression as well as the luciferase activity of a reporter construct containing the 3'-untranslated region of K-Ras messenger RNA. Furthermore, the miR-18a* repression also increased the cell proliferation and promoted the anchorage-independent growth in soft agar of human squamous carcinoma A431 cells, colon adenocarcinoma HT-29 cells and fetal hepatic WRL-68 cells. On the other hand, ectopic expression of miR-18a* by transfection with miR-18a* precursor suppressed K-Ras expression, cell proliferation and anchorage-independent growth of A431 cells. The increase in cell proliferation and anchorage-independent growth upon miR-18a* repression was, however, rendered by the Ras inhibitor farnesylthiosalicylic acid. In conclusion, miR-18a* may function as a tumor suppressor by targeting on K-Ras. Therefore, the miRNA may also be a potential therapeutic agent or target for cancer therapy.

Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed.

Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P0.05-0.001) and induced cell death (3-51%, P0.01-0.001) in a dose (5-75 microM)- and time (12-72 h)-dependent manner, which was associated with an increase in G(1) arrest. G(0)/G(1) phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin kinase inhibitors (Cdki) and cyclins. Our western blot analysis showed that berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P0.05-0.001) than non-berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

Although the precise role for the tuberous sclerosis complex-2 tumor suppressor gene (Tsc2) in tumor suppression is not clear, many studies have implicated Tsc2 in the regulation of cell differentiation, cell cycle control, GTPase activity, transcription, polycystin-1 localization, and translation initiation. We propose that Tsc2 also increases susceptibility to apoptosis, and that this functional role may contribute to the tumor suppressor activity of Tsc2. We previously characterized the apoptotic response of a Tsc2-null renal tumor cell line (ERC-18) to the tumor promoter okadaic acid (OKA). In the present study, we expressed Tsc2 in ERC-18 cells and compared the effect of Tsc2 expression on apoptotic induction. Tsc2 expression increased the susceptibility of ERC-18 cells to apoptosis induced by OKA and the phosphatidylinositol-3' kinase inhibitor, LY294002. In addition, Tsc2 expression abrogated OKA-induced cell detachment of ERC-18 cells. These results indicate that the OKA-induced, caspase-independent detachment previously observed in ERC-18 cells is Tsc2-dependent, and may support an additional role for the Tsc2 in regulating cell adhesion.

alpha-Santalol, an active component of sandalwood oil, has been studied in detail in recent years for its skin cancer preventive efficacy in murine models of skin carcinogenesis; however, the mechanism of its efficacy is not defined. Two major biological events responsible for the clonal expansion of transformed/initiated cells into tumors are uncontrolled growth and loss of apoptotic death. Accordingly, in the present study, employing human epidermoid carcinoma A431 cells, we assessed whether alpha-santalol causes cell growth inhibition and/or cell death by apoptosis. Treatment of cells with alpha-santalol at concentrations of 25-75 microM resulted in a concentration- and a time-dependent decrease in cell number, which was largely due to cell death. Fluorescence-activated cell sorting analysis of Annexin V/propidium iodide (PI) stained cells revealed that alpha-santalol induces a strong apoptosis as early as 3 h post-treatment, which increases further in a concentration- and a time-dependent manner up to 12 h. Mechanistic studies showed an involvement of caspase-3 activation and poly(ADP-ribose) polymerase cleavage through activation of upstream caspase-8 and -9. Further, the treatment of cells with alpha-santalol also led to disruption of the mitochondrial membrane potential and cytochrome c release into the cytosol, thereby implicating the involvement of the mitochondrial pathway. Pre-treatment of cells with caspase-8 or -9 inhibitor, pan caspase inhibitor or cycloheximide totally blocked alpha-santalol-caused caspase-3 activity and cleavage, but only partially reversed apoptotic cell death. This suggests involvement of both caspase-dependent and -independent pathways, at least under caspase inhibiting conditions, in alpha-santalol-caused apoptosis. Together, this study for the first time identifies the apoptotic effect of alpha-santalol, and defines the mechanism of apoptotic cascade activated by this agent in A431 cells, which might be contributing to its overall cancer preventive efficacy in mouse skin cancer models.

Gangliosides have been described as modulators of growth factor receptor activity and subsequent cellular function. Due to the lower-pH environment found in tumor cells, ganglosides are thought to be formed (at least to some extent) into their lactone forms. The aim of the study was to analyze the mode of action of the lactone of the ganglioside GM3 on epidermal growth factor (EGF) signaling in human ovarial epidermoid carcinoma A431 cells and cell growth in human oral epidermoid carcinoma KB cells by applying the GM3 lactone analog HK1-ceramide 2, which is stable under hydrolytic conditions. Specific inhibition of EGF-dependent receptor tyrosine phosphorylation was observed by HK1-ceramide 2 at 25 microM, whereas GM3 showed a comparable inhibition at eightfold higher concentrations. In cells exposed to low pH, where GM3 is thought to form its lactone to a higher extent, addition of GM3 showed no further inhibitory effect on EGF-dependent receptor phosphorylation. Similarly to GM3, HK1-ceramide 2 does not affect binding of (125)I-EGF to the cell surface receptor. EGF-dependent growth of KB cells was also found to be inhibited by HK1-ceramide 2 at much lower concentrations compared to GM3. In conclusion, our results indicate that the GM3 lactone analog HK1-ceramide 2 is a specific inhibitor of EGF receptor function and is more potent in reducing EGF-dependent tyrosine phosphorylation of the receptor in A431 cells and in inhibiting EGF-dependent growth of KB cells compared to GM3.

Gangliosides are able to bind to the epidermal growth factor receptor and inhibit its activation, but the mechanism of this inhibition is unknown. To address the role of receptor carbohydrates in facilitating interaction with gangliosides, we examined the ability of GM3 to bind the deglycosylated receptor and inhibit its autophosphorylation. Flow cytometry studies demonstrated that deglycosylation of the receptor did not affect its ability to be transported to the cell membrane. In contrast with the native (fully glycosylated) receptor, GM3 did not coimmunoprecipitate with the deglycosylated receptor. Using a novel colorimetric bead binding assay, GM3 was shown to bind well to the immunoprecipitated native receptor but not at all to the deglycosylated receptor. Finally, the addition of GM3 to cells with deglycosylated epidermal growth factor receptors did not result in significant further inhibition of autophosphorylation of the receptor, despite a 10-fold decrease in phosphorylation of the native epidermal growth factor receptor by 200 microM GM3. These studies suggest that ganglioside affects epidermal growth factor receptor activity through a direct interaction that requires receptor glycosylation, and contribute to our understanding of the role of gangliosides in cell membrane function.

A431 cells/UVC-induced apoptosis/Caspase 8/Fas/JNK/PAPK. We previously observed that p53-mutated human epithelial tumor A431 cells underwent apoptosis after ultraviolet C (UVC) irradiation through the caspases-8 and -3 pathway. Fas/FasL is known to initiate apoptosis in several cell lines via caspase-8 activation. Then, to determine if Fas/FasL mediates apoptosis in A431. we investigated Fas expression and modulation in UVC-irradiated A431 cells. A431 constitutively expressed Fas, which gradually decreased after UVC-irradiation. Pretreatment with a neutralizing anti-Fas antibody, ZB4, did not abrogate the UVC-induced apoptosis. An agonistic anti-Fas antibody, CH11, very slowly induced apoptosis in A431. suggesting that the constitutively expressed Fas had a low functional potential. Hence, UVC-induced apoptosis in A431 seems to occur independent of the Fas signal. Interestingly, however, a pretreatment with CH11 remarkably potentiated UVC-induced apoptosis. An inhibitor of caspase-8, Ac-IETD-CHO, partially inhibited UVC-induced apoptosis. JNK was phosphorylated immediately after exposure to UVC. prior to apoptotic chromatin condensation. Our data suggest that the activation of caspase-8 occurs independent of Fas upregulation, and that JNK/ SAPK contributes to UVC-induced apoptosis in human epithelial A431 cells.

CD40 is a 50 kDa molecule, a member of the tumor necrosis factor/nerve growth factor receptor family. It is expressed on B cells, monocytes, dendritic cells and various malignant cells. While the critical relevance of this molecule in T cell-dependent B cell activation is already established, the biological role of CD40-CD154 interaction in non-hematopoietic cells is still unknown. Here we show that CD40 is functionally expressed on human melanoma-derived cell lines. No correlation between surface CD40 expression and the origin of the cell line, primary versus metastatic, was observed. Melanoma cells were shown to be able to co-stimulate TCR-triggered human T cells; moreover, because they do not express CD80 or CD86 co-stimulatory structures, the involvement of additional pathways have to be postulated. We have identified CD40 as one of the molecules involved in melanoma cell-mediated co-stimulation of anti-CD3-triggered human CD4(+) T lymphocytes. In addition, a CD40-dependent pathway, able to enhance tumor cell proliferation at low serum concentrations, in vitro, has been shown to be functional in human melanoma cell lines.

The function of the pre-B cell receptor (pre-BCR) during B cell differentiation is not precisely defined. To investigate the pre-BCR receptor activity, we have established pre-BCR-positive pre-B cell lines that are able to differentiate into immature B cells in vitro. Antibody cross-linking of the pre-BCR induced apoptosis and differentiation accompanied with tyrosine phosphorylation. A specific tyrosine-phosphorylated 43 kDa protein (p43) was found down-stream of the pre-BCR. The results demonstrated the receptor function of pre-BCR, which indicates that a ligand-like molecule or a cross-linking structure on the cell surface is possibly present.

Inadequate cellular compartmentalisation of plasmid DNA and antisense oligodeoxynucleotides (ODNs) is generally considered as a major limitation in their use. In this study, an approach combining in situ visual-isation of rhodamine-labelled ODNs and affinity modification of proteins by radiolabelled-alkylating ODN derivatives has been used to investigate the uptake of ODNs into keratinocytes. We confirm here that unmodified ODNs are efficiently taken up and accumulate in cell nuclei in primary keratinocytes as well as in HaCaT and A431 keratinocyte cell lines. Uptake is fast, irreversible, saturable and not significantly altered by incubation at low temperature. Affinity modification studies in keratinocyte cell lines has revealed two high-affinity, cell-specific interactions between ODNs and proteins of 61-63 kDa and 35 kDa. Trypsin pre-treatment of A431 cells and pre-incubation with polyanions, or with unlabelled nucleic acid competitors, inhibited the accumulation of rhodamine-labelled ODNs in nuclei as well as the affinity labelling of the 61-63 kDa doublet and 35 kDa ODN-binding proteins by reactive ODN derivatives. Finally, cell fractionation studies indicated that these ODN-binding proteins were essentially localised in the plasma membrane. Our results suggest that these ODN-binding proteins might be involved in the recognition and transport of ODNs into keratinocytes.

We have developed a multistage model system in which a normal mouse keratinocyte clone has been initiated with 7,12-dimethylbenz[a]anthracene and variant clones derived with benign or malignant phenotypes. To identify specific genes altered during mouse skin carcinogenesis, the gene expression patterns of the normal parental epidermal cell, an initiated cell, a benign papilloma, and a poorly differentiated squamous cell carcinoma were compared using RNA differential display. Most alterations in gene expression were observed at malignant conversion, that is, in the poorly differentiated squamous cell carcinoma that is known to have deregulated expression of p53. The sequence of a cloned cDNA fragment lost in the poorly differentiated squamous cell carcinoma was nearly identical to the 3' region of an adhesion-related kinase which is involved in homophilic cell aggregation. It is found in normal epidermal progenitor cells as well as tumorigenic cells with differentiation potential, but not in tumorigenic cells with a poorly differentiated phenotype, suggesting that this adhesion-related kinase may be involved in epidermal cell differentiation. Differential display within the cloned epidermal cell model appears to be useful in detecting and identifying malignant conversion-associated genes which then can be tested directly for their potential role in epithelial carcinogenesis.

Background: The fragile histidine triad (FHIT) gene at chromosome 3p14.2 has been proposed to be a candidate tumor suppressor gene in human cancers. To test whether FHIT exhibits the functional properties of a tumor suppressor gene, we studied the expression of its protein (pFHIT) in human carcinoma cells and examined the ability of FHIT to inhibit the neoplastic phenotype of cancer cells. Methods: Subcellular localization and patterns of protein expression in tumor cells were determined by immunohistochemical analysis and immunoblotting with the use of polyclonal antipFHIT antisera. In tumor cells with undetectable pFHIT, we examined the effect of recombinant pFHIT expression on morphology, growth rate, colony formation, and in vivo tumor formation. Results: We demonstrated that pFHIT is a cytoplasmic 17-kd polypeptide whose expression could not be detected in 30 of 52 human carcinoma cell lines tested. We observed, however, that the stable overexpression of pFHIT did not alter cell morphology, inhibit colony formation, or inhibit cell proliferation in vitro. Furthermore, overexpression of pFHIT did not lead to altered cell cycle kinetics in dividing cells. The in vivo tumorigenicity of a tumor cell line that expressed high levels of recombinant pFHIT was equivalent to that of control transfectants and of parental cells. Conclusions: These results suggest that the replacement of pFHIT in human carcinoma cells does not suppress tumor cell growth and that this protein may be involved in tumorigenesis in ways that are distinct from the ‘‘classic’’ tumor suppressor paradigm. [J Natl Cancer Inst 1998;90: 426‐32]

Background and Purpose: The polyphenolic compounds present in green tea show cancer chemopreventive effects in many animal tumor models. Epidemiologic studies have also suggested that green tea consumption might be effective in the prevention of certain human cancers. We investigated the effect of green tea polyphenols and the major constituent, epigallocatechin-3-gallate, on the induction of apoptosis (programmed cell death) and regulation of cell cycle in human and mouse carcinoma cells. Methods: Human epidermoid carcinoma cells (cell line A431), human carcinoma keratinocyte (cell line HaCaT), human prostate carcinoma cells (cell line DU145), mouse lymphoma cells (cell line L5178Y), and normal human epidermal keratinocytes (NHEKs) were used. Apoptosis was assessed by 1) the formation of internucleosomal DNA fragments by agarose gel electrophoresis, 2) confocal microscopy, and 3) flow cytometry after tagging the DNA fragments by fluorescence label. The distribution of cells in different phases of the cell cycle was analyzed by flow cytometry. Results: Treatment of A431 cells with green tea polyphenols and its components, epigallocatechin-3-gallate, epigallocatechin, and epicatechin-3-gallate, resulted in the formation of internucleo-somal DNA fragments, characteristic of apoptosis. Treatment with epigallocatechin-3-gallate also resulted in apoptosis in HaCaT, L5178Y, and DU145 cells, but not in NHEK. Confocal microscopy and flow cytometry confirmed the findings. The DNA cell cycle analysis showed that in A431 cells, epigallocatechin-3-gallate treatment resulted in arrest in the G 0 -G 1 phase of the cell cycle and a dose-dependent apoptosis. Conclusions: Green tea may protect against cancer by causing cell cycle arrest and inducing apoptosis. It needs to be evaluated in human trials.

Background Triple-negative breast cancers (TNBCs) are typically more aggressive and result in poorer outcomes than other breast cancers because treatment options are limited due to lack of hormone receptors or amplified human epidermal growth factor receptor 2 (HER2). Many TNBCs overexpress the epidermal growth factor receptor (EGFR) or manifest amplification of theEGFRgene, supporting EGFR as a therapeutic target. While EGFR-directed small molecule inhibitors have shown limited effectiveness in clinical settings, use of EGFR as a mechanism of delivering enzymatic cytotoxins to TNBC has not been demonstrated. Methods Using the single-chain variable fragment (scFv) of the 806 antibody that binds only cells with overexpressed, misfolded, or mutant variants of the EGFR, a recombinant immunotoxin was engineered through gene fusion withPseudomonas aeruginosaExotoxin A (806-PE38). The potency of 806-PE38 on reducing TNBC cell growth in vitro and in xenograft models (n ≥ 6) was examined for six TNBC cell lines. All statistical tests were two-sided. Results 806-PE38 statistically significantly reduced the viability of all tested TNBC lines, with IC50values below 10 ng/mL for three of six cell lines, while not affecting cells with wild-type EGFR (IC50>300 ng/mL). Systemic treatments with 806-PE38 vs vehicle resulted in statistically significantly reduced tumor burdens (806-PE38 mean = 128 mm(3)[SD = 46 mm(3)] vs vehicle mean = 749 mm(3)[SD = 395 mm(3)], P = .001) and increased median survival (806-PE38 median = 82 days vs vehicle median = 50 days,P= .01) in a MDA-MB-468 TNBC mouse xenograft. Deletion of the catalytic residue eliminated both cytotoxic activity in vitro and the reduction in tumor burden and survival (P= .52). Conclusions These data support the further development of the 806-PE38 immunotoxin as a therapeutic agent for the treatment of patients with EGFR-positive TNBC. Follow-up experiments with combination therapies will be attempted to achieve full remissions.

The expression of N-acetylglucosaminyltransferase V (Glc-NAc-T V) is increased in many oncogenically transformed cells and in cell lines which are induced to proliferate. In order to characterize the regulation of GlcNAc-T V at the molecular level, we have examined the expression of Glc-NAc-T V mRNAs in a number of mouse tissues and in several human and rodent cell lines. The GlcNAc-T V mRNA is expressed in different amounts in the various mouse tissues, with the greatest amount observed in brain, followed by thymus, kidney, lung, intestine, heart and stomach, and no transcripts detected in liver or skeletal muscle. Measurements of GlcNAc-T V enzymatic activity, by contrast, show brain to have lower levels of activity than several of the other tissues, suggesting possible post-translational regulation. We find that there are two GlcNAc-T V transcripts in most of the RNAs analysed. The rodent cell lines all express both a 7.5 and a 9.5 kb mRNA, with the smaller transcript being more abundant. The human cells have mRNAs of 4.5 and 9.5 kb. Both mRNAs are expressed in HepG2 and MCF-7 cells, while A431 cells express only the 4.5 kb mRNA and HL60 cells express only the 9.5 kb transcript. Furthermore, only the 9.5 kb mRNA appears to be increased, along with activity, when HepG2 cells are stimulated to proliferate, suggesting that the two mRNAs may be under different regulatory controls. Also, a GlcNAc-T V-deficient, mutant lymphoma cell line, PHAR2.1, was found to express mRNAs which are larger than the normal mRNAs, possible due to an insertion or aberrant splicing, resulting in a defective mRNA.

Inostamycin, a novel microbial secondary metabolite, inhibited [3H]inositol and 32P1 incorporation into phosphatidylinositol (PtdIns) induced by epidermal growth factor (EGF) in cultured A431 cells, the IC50 being 0.5 micrograms/ml, without inhibiting macromolecular synthesis. The drug inhibited cellular inositol phosphate formation only when it was added at the same time as labeled inositol. It was found to inhibit in vitro CDP-DG:inositol transferase activity of the A431 cell membrane, the IC50 being about 0.02 micrograms/ml. It did not inhibit tyrosine kinase, PtdIns phospholipase C, or PtdIns kinase. Therefore, inhibition of PtdIns turnover by inostamycin must be due to the inhibition of CDP-DG:inositol transferase. Thus, inostamycin is a novel inhibitor of CDP-DG:inositol transferase.

The distribution of the epidermal growth factor receptor (EGFR) in mouse testis was ascertained by immunocytochemical methodology using a polyclonal antibody (RK2) shown previously to recognize the cytoplasmic domain of the human (A431 cells), murine (Swiss 3T3 cells), and chicken (CK 109 cells) EGFR. Initial studies performed to determine the usefulness of this antibody as a probe of the murine EGFR in testis employed two murine cell lines, TM4 and MA10, of Sertoli cell and Leydig cell origin, respectively, in which a physiological response of EGF and specific binding of iodinated EGF has been demonstrated. Western blotting in membrane preparations of TM4 and MA10 revealed only one prominent band at 170 kDa. Immunocytochemical localization in TM4 and MA10 cells illustrated a plasma membrane distribution of the receptor. Western blotting of membrane fractions prepared from testis also revealed a specific band at 170 kDa. In the intact testis, the EGFR was immunolocalized specifically in Leydig cells and Sertoli cells only. These results suggest that the involvement of EGF action in spermatogenesis may occur at the level of the somatic components of the testes, principally in the Leydig and Sertoli cells.

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) caused partial inhibition of the binding of [125I]epidermal growth factor ([125I]EGF) to intact mouse epidermal cells (HEL/37). The proportion of [125I]EGF binding which was insensitive to TPA inhibition decreased with increasing EGF concentration. The partial inhibition of [125I]EGF binding was not due to destruction of TPA or to covalent linkage of EGF to receptor sites. The binding of [125I]EGF to HEL/37 cells showed a curvilinear Scatchard plot; this was converted into a linear plot in the presence of TPA. We conclude that TPA acts to inhibit interactions between EGF receptors or between EGF receptors and some other membrane component(s).

The effect of human epidermal growth factor (hEGF) on the growth of various histological types of six human gastric carcinoma cell lines was examined. The cell lines had relatively high affinity EGF receptors (dissociation constant Kd = 10(-9) to 10(-10) M). One gastric cancer cell line, MKN-74 (well differentiated adenocarcinoma) showed no response to hEGF, in cell growth, DNA synthesis or 125I-hEGF cell binding. There were no apparent correlations between histological type and cell growth, DNA synthesis or number of EGF receptors in these cells. The number of EGF receptors and the Kd value of the gastric carcinoma cell lines varied with their internal and external environments. hEGF concentrations corresponding to maximum stimulation in DNA synthesis varied between cell lines. The results suggest some gastric carcinoma cells to have EGF receptors and their growth seemingly to be stimulated by EGF in vitro. There are, however, no obvious correlations between the effect of hEGF on the growth of human gastric carcinoma cell lines or their histological type.

Epidermal growth factor (EGF) receptor biosynthesis was examined in an oral squamous cell carcinoma line, NA, which overproduces the receptor to an even greater extent than the widely studied A431 cells. The EGF receptor of NA cells synthesized in the presence of tunicamycin had an apparent molecular weight of 130,000. The nascent protein in untreated cells was cotranslationally glycosylated to Mr 160,000 and further processed to Mr 170,000. The endo-beta-N-acetylglucosaminidase H (Endo H) digestion analysis revealed the presence of high mannose type oligosaccharide on the Mr 170,000 mature receptor. We extended the analysis by correlating the biosynthesis with the acquisition of binding activity. The unglycosylated Mr 130,000 receptor and the Mr 160,000 receptor seen after pulse-labeling had no EGF binding activity, whereas the Mr 160,000 receptor seen after chase-incubation and the Mr 170,000 receptor had binding activity. Thus, not only glycosylation but also some oligosaccharide processing is apparently necessary for the EGF binding. Treatment with processing inhibitors, such as monensin, swainsonine and 1-deoxynojirimycin, affected neither receptor transport to the plasma membrane nor binding activity. Inhibition by 1-deoxynojirimycin is thought to be incomplete since the surface receptor in treated cells had the same molecular weight as that in control cells. An Mr 160,000 receptor without binding activity accumulated in the intracellular fraction in the presence of brefeldin A, an inhibitor of intracellular transport. Thus, the EGF binding activity is thought to be acquired after the brefeldin A-sensitive process but prior to the swainsonine-sensitive mannose removal in NA cells.

Using tumour cell lines derived from human bone tumours, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator in many tissues, and its effect on synthesis of prostaglandin (PG) E2, a potent bone-resorbing factor, by cultured osteosarcoma cell line were studied. Three tumour cell lines, one osteosarcoma (HOSO) and two giant cell tumours of the bone (G-1 and G-2), all possessed specific binding sites for 125I-labelled EGF: the apparent dissociation constant was ~4–10 × 10−10 m and the maximal binding capacity was 50 000–80 000 sites/cell. EGF had no mitogenic effect in these cell lines. However, these cell lines did not have specific binding sites for 125I-labelled parathyroid hormone (PTH) or calcitonin. HOSO line produced and secreted PGE2 into medium, while no significant amount of PGE2 was demonstrated in G-1 or G-2 line. EGF significantly stimulated PGE2 production in HOSO line in a dose-dependent manner (0.5–50 ng/ml); its stimulatory effect was completely abolished by indomethacin, an inhibitor of PG biosynthesis. Exogenous PGE1 significantly stimulated cyclic AMP formation in HOSO line, whereas PGF2α, PTH, calcitonin, or EGF had no effect. None of these calcium-regulating hormones affected cyclic AMP generation in either G-1 or G-2 line. These data indicate that human bone tumour cells have specific EGF receptors unrelated to cell growth, and suggest that EGF may be involved in bone resorption through a PGE2-mediated process in human osseous tissues.

Monoclonal antibodies (MoAbs) were developed against epidermal growth factor (EGF) receptor on the human epidermoid carcinoma cell line A431. The A431 antigen recognized by the MoAbs has an apparent molecular weight of approximately 170,000, with the same molecular weight as the CNE-2 cell line (poorly differentiated nasopharyngeal carcinoma). Administration of anti-EGF receptor MoAbs inhibited tumor formation, caused by the CNE-2 and A431 cell lines, in athymic mice. When the same MoAbs were used in therapy against Tca8113 (a human tongue carcinoma) and HeLa cells (a human cervical carcinoma), tumor growth was not affected. The number of EGF receptors and the apparent dissociation constants for 125I-EGF on CNE-2 and A431 were 1.3 x 10(5)/cell (Kd 7.7 x 10(-8) M) and 1.4 x 10(6)/cell (Kd 2.4 x 10(-9) M), respectively. Three anti-EGF receptor MoAbs were used in these studies. MoAbs 3 and 176, capable of competing with EGF for receptor binding, showed significant tumor growth inhibition. MoAb 101 was incapable of blocking the binding of EGF to its receptor and was not as effective as MoAbs 3 and 176 in tumor growth inhibition. Our observation is that in vitro, MoAb anti-EGF receptor is cytostatic, rather than cytocidal, against CNE-2 and A431.

The pathogeneses of hypercalcemia and hypophosphatemia which developed in a patient with metastatic invasive ductal breast carcinoma were studied. The patient had low plasma levels of immunoreactive parathyroid hormone (PTH) and 1,25(OH)2D, increased nephrogenous cyclic adenosine monophosphate (cAMP) excretion and low TmPO4/GFR, suggesting the presence of humoral PTH-like activity. The tumor extract showed activities which would stimulate bone resorption in vitro and cAMP generation in the osteogenic cell line, MC3T3 E1, and in the rat kidney cortex. In addition, the extract stimulated epidermal growth factor (EGF)-independent colony formation of the NRK 49F cells in soft agar, and inhibited the binding of EGF to A431 cells, indicating it to have transforming growth factor (TGF)-alpha activity. The extract contained appreciable amounts of immunoreactive PTH-related protein (PTH-rP) but negligible amounts of immunoreactive PTH. Thus, the PTH-like activity for stimulating cAMP generation in the bone and kidney was attributed to PTH-rP. Chromatographic analyses on reverse phase high performance liquid chromatography (HPLC) separated the PTH-rP activity from that of TGF-alpha and the bone resorbing activity in vitro was found only in the fractions of PTH-rP. It was concluded that this breast cancer produced PTH-rP as well as TGF-alpha, and the former was thought to have a major role to play in the humoral hypercalcemia of malignancy observed in this patient.