Reversal of methotrexate resistance in human squamous carcinoma cells by SQM1-liposome.

SQM1, a membrane protein associated with methotrexate (MTX) transport was incorporated into unilamellar vesicles, forming. SQM1-Liposome. Human squamous carcinoma of the head and neck (SqCHN) cells resistant to MTX with defective MTX transport and low SQM1 expression were treated with SQM1-Liposome. SQM1 content was found to be increased in these treated SqCHN cells by radioimmunoassay. Concurrent increases in MTX uptake and MTX cytotoxicity in these treated SqCHN cells were found. The ability to reverse MTX-resistance in vitro opens further possibilities for in vivo applications.

Prognostic significance of early response to a single dose of asparaginase in childhood acute lymphoblastic leukemia.

PURPOSE: The in vitro and in vivo efficacy of a single dose of asparaginase in children with newly diagnosed acute lymphoblastic leukemia and the correlation between in vitro and in vivo antileukemic response and long-term outcome were prospectively evaluated. PATIENTS AND METHODS: Two hundred fifty-one patients were randomized to receive 1 of 3 asparaginase preparations (Escherichia coli, Erwinia chrysanthemi [Erwinia], or pegaspargase). In vitro assessment of efficacy was expressed as the percent total cell kill (TCK), based on the number of viable cells found after 5 days of culture in the presence of asparaginase. In vivo leukemia cell kill (LCK) was calculated by comparing bone marrow cellularity and percent leukemic blasts in marrow obtained before and 5 days after treatment with a single dose of asparaginase. Acute toxicity was determined by clinical and laboratory assessment. RESULTS: There was equivalent cell kill with all three types of asparaginase. The mean in vitro TCKs for E. coli, Erwinia, and pegaspargase were 31%, 39%, and 36%, respectively (P = 0.63). The mean LCKs in marrow of patients exposed to E. coli, Erwinia, and pegaspargase were 69%, 74%, and 65%, respectively (P = 0.88). The lack of response to asparaginase in vitro predicted a higher risk for clinical relapse regardless of risk assignment (12 leukemic events among 21 in vitro nonresponders; 57%, P < 0.001). There was no difference in acute toxicity among the three asparaginase preparations. CONCLUSIONS: All three asparaginase preparations produced equivalent LCKs in in vitro and in vivo analyses. In vitro response to asparaginase provided a risk group-independent prognostic factor.

A new membrane marker for commitment of human bronchial epithelial cells to terminal squamous differentiation.

Squamous differentiation in human bronchial epithelial (HBE) cells is a metaplastic change that occurs primarily after chronic injury and often precedes squamous cell carcinoma. After treating HBE cells with inducing agents for various periods followed by removal of these agents and assay for terminal squamous differentiation, HBE cells were found to become committed sqamous differentiation after 12-36 hours of treatment. Expression of a membrane marker, SQM1 protein, increased during the commitment stage, peaked shortly after commitment and gradually decreased in the later stages of squamous differentiation of HBE cells. The lack of marked morphological and biochemical changes associated with the commitment and early stages of squamous differentiation makes SQM1 protein a useful marker for the study of squamous differentiation and metaplasia of HBE cells.

Congruence of SQM1 protein expression with methotrexate sensitivity and transport.

Expression of SQM1 protein, a membrane protein, was shown to be reduced in human squamous carcinoma of the head and neck (SqCHN) cell lines made less sensitive to methotrexate (MTX). High correlation of SQM1 protein expression with MTX transport (r = .94) and MTX sensitivity (r = .94) was found in MTX-resistant sublines developed from a clonally drived SqCHN cell line, SCC 15S1. Unlike SQM1 protein, there was no significant difference in amount or molecular weight of reduced-folate binding protein found in the membrane of a MTX-resistant subline, SCC15R1-3 and SCC15S1. Furthermore, MTX surface membrane binding was not significantly altered in SCC15R1-3. Compared to SCC15S1 parent cell line, SCC15R1-3 subline with similar DHFR enzyme level and MTX polyglutamylation showed a marked reduction in MTX uptake due to a decrease in Vmax without a significant change in Kt. These findings suggest the existence of membrane molecules like SQM1 protein that may indirectly affect MTX transport.

Multiple NF-I-site binding factors associated with different cell-types of human lung carcinomas.

Nuclear extracts from human lung adenocarcinoma and squamous carcinoma cells were found to form two types of DNA-protein complexes with a consensus nuclear factor I (NF-I)-binding site. Three NF-I-site-binding cofactors (NCF90, NCFIIO and NCF120) were found to be differentially expressed in these two cell-types of human lung carcinomas. These data suggest that the NF-I-binding enhancer element with its differentially expressed NF-I cofactors may play an important role in the differentiation of neoplastic human bronchial epithelium.

Expression of enhancer binding factors associated with various cell types of lung cancer.

Differential expression of nuclear factors binding specifically to AP-1, AP-2, AP-3, and NF-I/CTF enhancer elements was found in the various cell types of human lung carcinoma cells. Adenocarcinoma and squamous carcinoma cells seemed to express higher levels of these factors than large-cell carcinoma and small-cell carcinoma cells. Among small-cell carcinoma cells, variant small-cell carcinoma cells which presumably are closer than classical small-cell carcinoma cells to non-small-cell carcinoma cells in terms of differentiation characteristics also expressed higher levels of these factors. Furthermore, nuclear extracts from the various cell types of carcinoma cells were found to produce different patterns of electrophoretic mobility shift even with the same enhancer element, suggesting the presence of multiple species of specific enhancer-binding activities in these cells. Thus, these enhancer elements may be used as probes for cell-type specificity in the study of differentiation of lung carcinomas.

Expression of a new surface membrane antigen (SQM1) in tumor cells cultured in media with different calcium ion levels.

Terminal differentiation is usually achieved in normal as well as transformed squamous epithelial cells when cultured. On the other hand, tumor cells at various differentiation stages and with different biological characteristics comprise the heterogeneous properties of tumors which have been one of the barriers to effective treatments. Recently, a surface membrane protein has been reported in squamous cell carcinomas of the head and neck, which is recognized by a murine monoclonal antibody, SQM1. This glycoprotein was further suggested to be related to squamous cell differentiation and intercellular adhesion. In a recent study, the esophageal carcinoma cells of EC/CUHK2 cell line were induced to various differentiation stages as evidenced by the increasing amount of intracellular desmosomes and tonofilaments and greater binding ratios of cytokeratin and involucrin antibodies than in those cells that maintained lower calcium ion concentrations. The expression of SQM1 antigen was found to increase in intensity when the tumor cells were cultured in moderate to high calcium ion levels for 10 to 15 hours when the differentiation patterns were beginning to appear. The intensity declined gradually thereafter. Thus SQM1 protein might be related to the stage when the cells started committing with squamous differentiation.

Reduced membrane protein associated with resistance of human squamous carcinoma cells to methotrexate and cis-platinum.

A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SqCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SqCHN cells which developed resistance to the alkylating drug cis-latinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors.

Differential utilization of the SV40 enhancer and early promoter in various human lung carcinoma cell lines.

Cell lines derived from the major morphological types of human lung cancer were tested for their ability to utilize the SV40 enhancer/early promoter. These cell lines were transfected with a recombinant plasmid containing a reporter gene, coding for chloramphenicol acetyltransferase (CAT), under the control of the SV40 enhancer/early promoter. The transfected cells were then assayed for CAT activity. Non-small-cell carcinomas, especially squamous carcinomas, were found to be 2-3 orders of magnitude more efficient in utilizing the SV40 enhancer/early promoter than small-cell carcinomas. The presence of different SV40 enhancer/early promoter specific DNA-binding nuclear factors in squamous and small-cell carcinomas was demonstrated in gel mobility shift experiments. These observations seem to suggest that the set of transcriptional regulatory factors associated with squamous carcinomas may be distinct from that associated with small-cell carcinomas.

cDNA cloning of a novel cell adhesion protein expressed in human squamous carcinoma cells.

A novel protein (SQM1 protein) present in human squamous epithelial cells has been found to be involved in cell adhesion in squamous epithelial cells, endothelial cells and extracellular matrix proteins. The corresponding cDNA that encodes a 135-residue polypeptide has been isolated. Sequence analysis indicates that the encoded polypeptide is distinct yet related to the beta subunit of integrins. A new sequence motif consisting of a heptadic repeat of positively-charged residues present in the polypeptide has been identified and is proposed to be important in protein-protein interaction. These results suggest that SQM1 protein, a new cell adhesion molecule expressed in squamous epithelial cells, may play an important role in tumor metastasis.

Purging of small cell lung cancer-contaminated bone marrow by monoclonal antibodies and magnetic beads.

At diagnosis, most patients with small cell lung cancer (SCLC) have bone marrow involvement detected by immunological techniques. As preparative regimens become more effective in the elimination of systemic disease, bone marrow purging may become a significant intervention, even more so with the trend toward use of greater numbers of stem cells for reconstitution (such as peripheral blood stem cells). The optimal conditions for removal of SCLC cell lines from bone marrow mixtures with magnetic bead immunoconjugates are described. Metabolically labelled SCLC cells (SW2, OC2, NCI-H82) were incubated with mouse monoclonal antibodies reactive with SCLC membrane antigens (SM1, LAM2, LS1) alone or in combination. Magnetic beads conjugated with secondary goat anti-mouse-IgG or IgM antibody were added and the unpurged cells decanted off. Tumor removal was evaluated by residual radioactive counts in the decanted cell mixture as well as by clonogenic assay. The log removal was compared to controls performed without primary or secondary antibody. All assays were performed in triplicate. Bone marrow toxicity was similarly evaluated. In a simulation of bone marrow purging, a bead to total cell ratio of greater than or equal to 10:1 provided maximal tumor removal regardless of the degree of contamination. However, when the bead to total cell ratio dropped lower than 10:1, purging efficiency lessened despite maintenance of bead: tumor cell ratio of 100:1. A concentration of primary antibody ten-fold greater than saturation by indirect immunofluorescence provided optimal elimination. Using single exposures of antibody and beads, a mixture of all 3 MoAbs resulted in 2.4-2.6 log removal for SW2 and OC2. However, with NCI-H82, a cell line with low expression of these antibodies on the cell surface, maximal elimination was only 1.6 logs. Double bead exposure for 30 minutes each was more effective than single treatments for 30-60 minutes. No significant toxicity to bone marrow function was observed in parallel studies. Magnetic bead separation appears to be an efficient and nontoxic method to purge bone marrow of tumor contamination with SCLC.

Ultrastructural localization of a new surface membrane antigen (SQM1) related to squamous differentiation.

Normal squamous epithelial cells readily undergo terminal differentiation in culture and are commonly used in differentiation studies. Several intracellular markers of squamous differentiation such as keratin, involucrin, transglutaminase and cholesterol sulfate have been well-studied and described by other workers. We have recently reported a surface membrane antigen in squamous carcinoma of the head and neck antigen in squamous carcinoma of the head and neck which is recognized by a murine monoclonal antibody SQMI. In this paper, we present our studies on the ultrastructural localization of SQMI antigen in cultured squamous epithelial cells using gold-labelled antibody. The cells studied included both normal and cancer cells at different degrees of differentiation. Under both transmission and scanning electron microscopy examination, the SQMI antigen was localized at the membrane surface of cultured cells, particularly at sites of cell-cell interdigitation. No association with desmosomal structure was observed in any of the specimens examined. There was however an association of SQMI antigen with microvilli of cell membrane. No non-specific cytoplasmic localization of SQMI antigen was observed. The intensity of SQM1 antigen revealed by gold-labelling appeared to have a positive correlation with the degree of differentiation of the cells in culture.

In vitro and in vivo killing of acute lymphoblastic leukemia cells by L-asparaginase.

L-Asparaginase (ASNase) is a potent antileukemic enzyme routinely used in the treatment of children with acute lymphoblastic leukemia. As part of investigations of the biological activity of ASNase, we have developed techniques which measure the in vitro and in vivo cell killing ability of ASNase. To study the effect of ASNase on in vitro survival of primary lymphoblasts, bone marrow mononuclear cells obtained from untreated patients with acute lymphoblastic leukemia were cultured with and without ASNase. After 5 days, viable cells were counted using trypan blue exclusion to calculate total cell kill due to ASNase. Propidium iodide exclusion, leukemia cell surface antigens, and flow cytometry were used to determine leukemia cell kill due to ASNase. Comparison of leukemia cell kill and total cell kill showed a direct linear relationship (n = 24, r = 0.7), preferential killing of leukemia cells by ASNase (slope = 0.66), and that use of leukemia cell surface markers yielded a more accurate measurement of leukemia cell killing. ASNase at concentrations from 0.0001 to 0.1 IU/ml had equal effects on extent of leukemia cell killing (P = 0.3 to 0.7), suggesting the absence of a dose response at the ASNase concentrations tested. As a measure of the in vivo response to ASNase treatment, the number of viable bone marrow leukemia cells in the patient prior to and 5 days after treatment with ASNase was measured as the product of (% of rhodamine 123 fluorescent [viable] cells) x (absolute leukemic infiltrate). The change which occurred in the viable leukemic infiltrate was the same for patients whether they received 25,000 or 2,500 IU/m2 of ASNase as a single drug. There was a linear correlation (n = 8, r = 0.9) between in vivo and in vitro leukemia cell killing by ASNase. Thus, the in vitro assay described here can be used to predict in vivo sensitivity to ASNase in acute lymphoblastic leukemia.

Keratin subtypes of small cell lung cancer.

The molecular forms of keratin in small cell lung cancer (SCLC) cell lines and tumors were examined with antikeratin monoclonal antibodies. Immunostaining of SCLC by antikeratin antibody and examination by fluorescence microscopy indicates population heterogeneity in keratin content. Intensity of immunostaining is often weak. However, polyacrylamide gel electrophoresis and immunoblotting reproducibly demonstrate the presence of keratin and allow analysis of the keratin subtypes. The finding of keratin subtypes closely associated with the development of keratinizing epithelium (the 68 kDa basic keratin) in SCLC was unexpected in a tumor that is regarded as poorly differentiated. The cytoskeletal composition of SCLC suggests the presence of a heterogeneous population with a significant proportion of cells expressing highly differentiated epithelial properties.

Localization of radiolabeled SM1 monoclonal antibody to small-cell lung cancer tumors in mice.

Small-cell carcinoma (SCCL) is an aggressive type of lung cancer. Though it is usually responsive to therapy, be it chemotherapy or radiation, the majority of patients eventually relapse and overall prognosis is dismal. New forms of therapy are, therefore, needed. SM1 monoclonal antibody (MAb) was developed in our laboratory and demonstrated to be highly reactive against SCCL. I125 radiolabeled SM1 antibody was administered intravenously to nude mice bearing SCCL tumor xenografts. The mice were sacrificed, different tissues sampled and tested for uptake of radioactivity five days following antibody injection. There was over a 30 fold increase in localization of labeled antibody to the tumor as compared to muscle tissue. All organs tested showed an insignificant amount of MAb (p = 0.01) including the spleen, which had the highest normal tissue uptake in these experiments. These results demonstrate that SM1 MAb can be successfully targeted to SCCL xenografts. Its potential applications for imaging and therapy of SCCL in man are currently under investigation.

Effect of fluosol-DA/carbogen on etoposide/alkylating agent antitumor activity.

The tumor growth delay produced by the combination of etoposide with the alkylating agent CDDP or BCNU and Fluosol-DA with carbogen breathing in three model tumor systems was examined. The addition of Fluosol-DA to etoposide treatment increased tumor growth delay 2.8-fold, 3.3-fold and 2.2-fold in the FSaIIC fibrosarcoma, the Lewis lung carcinoma and the SW2 small-cell xenograft, respectively. In both the FSaIIC fibrosarcoma and the Lewis lung carcinoma the combination of etoposide treatment with CDDP produced an additive effect. When Fluosol-DA was added to this combination the tumor growth delay increased 1.9-fold and 1.4-fold in the FSaIIC fibrosarcoma and the Lewis lung carcinoma, respectively. Adding Fluosol-DA to a treatment regimen with etoposide and BCNU produced a 2.2-fold, 2.0-fold and 1.6-fold increase in the tumor growth delay of the FSaIIC fibrosarcoma, the Lewis lung carcinoma and the SW2 small-cell xenograft, respectively. The effect of these various treatment combinations on tumor cell survival was assessed in the FSaIIC fibrosarcoma. When the alkylating agents CDDP or BCNU were prepared in Fluosol-DA, there was an additional increase in tumor cell kill, so that with CDDP there was 2.1-fold and 4.7-fold increase in tumor cell kill and with BCNU there was 1.5-fold and 1.2-fold increase in tumor cell kill compared to the drug plus Fluosol-DA and the drug plus Fluosol-DA/carbogen breathing, respectively. The combination of etoposide and CDDP led to less than additive cell killing, and the combination of etoposide and BCNU appeared to be additive, as predicted by simple product summation, in all of the treatment conditions examined. Both etoposide + CDDP and etoposide + BCNU produced additive or less than additive toxicity to bone marrow as measured by CFU-GM.

Immunohistochemical identification of squamous carcinoma of the head and neck with monoclonal antibody SQM1.

Frozen tissue sections of biopsies from head and neck squamous cancer lesions were examined for immunohistochemical staining with a recently developed monoclonal antibody, designated as SQM1 antibody and directed against the surface membrane of squamous carcinoma cells. SQM1 antibody stained selectively squamous carcinoma, while normal mucosa and cells of the stroma were non-reactive. Positive staining of tumor was found in 33/35 specimens obtained from several major sites of the head and neck area and was observed in primary manifestations and lymph node metastases as well as in recurrences. The most consistent reactivity was seen with carcinomas of the tongue. Well differentiated squamous carcinomas contained a higher proportion of SQM1 positive tumor cells than poorly differentiated carcinomas. We suggest that the SQM1 antibody may aid in the immunohistochemical identification of squamous carcinoma of the head and neck area.

Cytotoxic murine monoclonal antibody LAM8 with specificity for human small cell carcinoma of the lung.

The reactivity of the murine immunoglobulin monoclonal antibody LAM8 directed against a membrane antigen of human small cell carcinoma (SCC) of the lung was investigated on human cell lines and tissues. Indirect immunofluorescence staining, radioimmunoassays, and cytotoxicity assays showed LAM8 antibody to selectively react with SCC but not with non-SCC lung cancer cell lines and extrapulmonary tumor cell lines. Unlike other SCC antibodies, including those we have previously described, highly preferential reactivity with SCC tissues was also demonstrated by immunoperoxidase staining of deparaffinized formalin-fixed tissue sections. Membrane and cytoplasmic staining was seen in of 9 of 12 SCC tissues. No significant staining was seen in non-SCC lung cancer and a wide range of other tumors, including mesothelioma and bronchial carcinoids. Significant LAM8 reactivity was also absent in normal tissues of all major organs. Few tumors and epithelial tissues, including bronchial epithelium had rare LAM8 positive cells which were always less than 2% of the entire cell population. In vitro treatment with antibody and human complement was highly cytotoxic to SCC cells, but had not effect on bone marrow progenitor cells. Immunoblotting of membrane extracts separated on sodium dodecyl sulfate-polyacrylamide gels showed the LAM8 antigen to have a band of an approximate molecular weight of 135,000 and a cluster of bands with approximate molecular weights of 90,000. This reactivity was lost after incubation of the extracts with periodate. LAM8 antibody shows a highly preferential reactivity with SCC cell lines and formalin-fixed paraffin-embedded SCC tissues and is selectively cytotoxic to cells expressing LAM8 antigen.

SQM1 antibody defines a surface membrane antigen in squamous carcinoma of the head and neck.

We produced murine monoclonal antibodies (MAbs) directed against the surface membrane of squamous-cell carcinoma of the head and neck (SCCHN). One antibody, SQM1, was determined by immunofluorescence and radioimmunoassay to be reactive with 13/13 SCCHN cell lines derived from different sites of the head and neck area. No binding reaction was observed with normal fibroblasts, red blood cells, nucleated bone-marrow cells or epithelial cells from normal oral mucosa. SQM1 reactivity was observed with primary cultures of normal epidermal and bronchial epithelial cells. Significant reactivity was found with 2/4 cell lines derived from small-cell carcinoma of the lung but little or no reactivity was found with other lung cancer cell lines. Various cell lines derived from other cancers including breast, colon, and ovarian carcinomas, melanoma, neuroblastoma and leukemia were generally unreactive. Seventeen out of 18 fresh frozen specimens of SCCHN were strongly reactive with SQM1 antibody. However, autopsy specimens from the heart, liver, kidney, spleen, colon, subcutaneous fat and skin connective tissue were unreactive. SQM1 antibody may be useful in biological and clinical studies of the head and neck region.