A rare polyadenylation signal mutation of the FOXP3 gene (AAUAAA-->AAUGAA) leads to the IPEX syndrome.

The mouse scurfy gene, Foxp3, and its human orthologue, FOXP3, which maps to Xp11.23-Xq13.3, were recently identified by positional cloning. Point mutations and microdeletions of the FOXP3 gene were found in the affected members of eight of nine families with IPEX (immune dysfunction, polyendocrinopathy, enteropathy, X-linked; OMIM 304930). We evaluated a pedigree with clinically typical IPEX in which mutations of the coding exons of FOXP3 were not detected. Our reevaluation of this pedigree identified an A-->G transition within the first polyadenylation signal (AAUAAA-->AAUGAA) after the stop codon. The next polyadenylation signal is not encountered for a further 5.1 kb. This transition was not detected in over 212 normal individuals (approximately 318 X chromosomes), excluding the possibility of a rare polymorphism. We suggest that this mutation is causal of IPEX in this family by a mechanism of nonspecific degradation of the FOXP3 gene message.

Rater agreement and utility of the mutagen-induced chromosome damage assay.

Chromosomal damage in peripheral blood lymphocytes induced by short-term in vitro exposure to the cytotoxic antibiotic bleomycin was first described in 1983 and proposed as a phenotypic assay for chromosome instability. This assay was subsequently described as potentially useful in assessing an individual's risk to environmental carcinogens in 1989. Since 1995 numerous published studies have used this assay to assess risk for cancer in the aerodigestive tract, particularly lung cancer, in various ethnic populations. Odds ratios up to 8.5 have been reported for individuals deemed "mutagen sensitive" (defined as > or = 1 chromatid break per metaphase averaged in 50 metaphases analyzed). While this phenotypic assay is appealing for lung cancer risk assessment it has not been reproduced by other investigators. Because of our interest in lung cancer biology, epidemiology, and genetics, we sought to independently assess the rater agreement of this assay. We found that 1) the assay is laborious to conduct (8 hours of labor) and relatively expensive (> $100), yet reducing the number of metaphases from 50 to 20 produced a reliable, less expensive, and less laborious test; and 2) the rater agreement of individual metaphase readings is poor, but agreement for a summary measure is high.

Evidence for genetic heterogeneity in hereditary neuralgic amyotrophy.

Hereditary neuralgic amyotrophy (HNA) is a rare autosomal dominant disorder characterized by recurrent episodes of severe arm and shoulder pain with weakness, atrophy, and sensory impairment in a brachial plexus distribution. Recent studies mapped the HNA locus to chromosome 17q25. Two pedigrees with clinically typical HNA in which markers from chromosome 17q25 do not cosegregate with the disease and in which lod scores do not support linkage to chromosome 17q25 were identified.

Synergistic interaction between anti-p185HER-2 ricin A chain immunotoxins and radionuclide conjugates for inhibiting growth of ovarian and breast cancer cells that overexpress HER-2.

Radionuclide conjugates or ricin A chain (RTA) immunotoxins that target pl85HER-2 have partially inhibited the growth of human ovarian cancer xenografts in athymic mice but generally have not cured mice bearing human tumor transplants. The present study was undertaken to explore whether a combination of ionizing radiation and an immunotoxin could exert additive or synergistic cytotoxicity in culture and in vivo against cancer cells that overexpress p185HER-2. In cell culture, treatment with 200-2000 cGy external beam irradiation followed by incubation with TA1-anti-pl85mHER2-RTA immunotoxin (TA1-RTA) produced synergistic inhibition of clonogenic growth of ovarian and breast cancer cells that expressed > 10(6) pl85HER-2 receptors/cell. The effect on cell survival correlated with an inhibition of DNA repair. A prior study (F. J. Xu et al, Nucl. Med. Biol., 24: 451-460, 1997) compared the biodistribution of radionuclide conjugates prepared with monoclonal antibodies that bind to different epitopes on the extracellular domain of pl85HER-2 and found optimal tumor uptake with the 520C9 antibody, which did not compete with TA1 for binding to the receptor. In this report, the TA1-RTA immunotoxin and the 131I-labeled 520C9 radionuclide conjugate could each inhibit the growth of clone-9002-18 xenografts in athymic mice but did not yield long-term survivors using maximally tolerated doses of each agent. When TA1-RTA and 131I-labeled 520C9 were used in combination, a greater inhibition of tumor growth was obtained than with either single agent. Similarly, survival with the combined treatment was significantly prolonged (P = 0.004) relative to treatment with immunotoxin or radionuclide conjugate alone. After treatment with an optimal combination of immunotoxin and radionuclide conjugate, 50% of mice survived >300 days, whereas controls succumbed with a median survival of 36 days. These results suggest that combinations of immunotoxins and radionuclide conjugates deserve further evaluation for the treatment of cancers that overexpress pl85HER-2.

SSA/RO52gene and expressed sequence tags in an 85 kb region of chromosome segment 11p15.5.

Frequent allelic loss in lung cancer has been described in a region on chromosome segment 11p15.5 (LOH11A). The region is approximately 650 kb in size and flanked by the markers D11S988 centromeric and D11S860 telomeric. Clinical and cell biological studies suggest that it contains a gene associated with metastatic tumor spread. One of the genes identified within this region is SSA/Ro52, which has a RING finger domain and may be involved in gene regulation. We studied this gene for mutations using SSCP analysis and for expression using RT-PCR and Western blotting on lung cancer cell lines and tumor-normal tissue pairs. No mutations and no differences in mRNA or protein expression between tumor tissue and normal tissue pairs were identified. We discovered a novel polymorphic site (SSA44C/T) within exon 1 of this gene. Among 141 primary lung cancers, allelic loss was observed in 16% of informative cases. Our analyses excluded SSA/Ro52 as a tumor-suppressor gene in lung cancer and newly defined the centromeric border of the LOH11A region from D11S988 previously to SSA44C/T. This reduced the region of the putative suppressor gene to 460 to 485 kb. A significant difference (p = 0.01) in the frequency of alleles for this polymorphism between Caucasians and African-Americans was observed. The "T" allele frequency was 0.12 in Caucasians and 0.23 in African-Americans. A genomic EcoRI map over 85 kb surrounding the SSA/Ro52 gene was constructed, and 4 expressed sequence tags were identified by sequencing and studied.

Maximizing the motivational impact of feedback of lung cancer susceptibility on smokers' desire to quit.

This two-by-two factorially designed study evaluate approaches for communicating feedback of lung cancer susceptibility to smokers as a method for motivating smoking cessation. The study factors were: method of communicating feedback (by mail with telephone follow-up or in-person) and carbon monoxide feedback (yes or no). One-hundred-forty-four smokers were stratified on race and randomized to one of four conditions. Participants were surveyed at baseline and 2-month follow-up. Polymerase chain reaction (PCR) testing for the absence of the glutathione S transferase mu (GSTM1) gene was the susceptibility marker. Regardless of counseling method or carbon monoxide (CO) feedback, the majority (90%) of smokers accurately recalled the test result and 66% accurately interpreted the meaning of the test result. Smokers who received their result in person were significantly less likely to have read the result booklet than those in the telephone counseling group (OR = .28, 95%; CI .12-.62; p < .05). Neither counseling method nor CO feedback increased smokers' perceived risks for lung cancer. However, at the counseling session those who received in-person counseling were significantly less frightened by the test result than those who received telephone counseling (OR = .42, 95%; CI .20-86; p < .05) and at the 2-month follow-up those who received a CO test were significantly less frightened by their susceptibility result (OR = .40, 95%; CI .17-.92; p < .05) than those who did not have a CO test. Evaluation of further refinements in communicating the meaning of susceptibility results to motivate smoking cessation is warranted.

Lung cancer and the human gene for ribonucleotide reductase subunit M1 (RRM1).

LOH11A is a region of Chromosome (Chr) 11p15.5 where 75% of lung cancers show loss of heterozygosity (LOH). Clinical and cell biological studies suggest that LOH11A contains a tumor/metastasis suppressor gene. We have mapped this region (650 kb) using overlapping genomic P1/PAC/BAC clones, and one of the genes that we have identified is RRM1. This gene encodes the large subunit (M1) of ribonucleotide reductase, the heterodimeric enzyme that catalyzes the rate-limiting step in deoxyribonucleotide synthesis. By comparing our genomic sequences with the previously published cDNA, we have found that the human gene is composed of 19 exons. It is oriented telomere to centromere and is Alu rich. In order to verify that RRM1 maps within the boundaries of LOH11A, we assessed the frequency of LOH at a SacI polymorphism within intron IX of the gene. We observed LOH in 48% (15/31) of informative lung tumor specimens. To determine whether RRM1 was mutated in tumors, SSCP analysis of the 19 RRM1 exons was performed. No mutations were revealed in 12 pairs of normal and tumor DNA samples. Immunoblots on protein extracts from normal/tumor pairs indicated that a protein of the expected size was present in both. Our conclusion is that RRM1 lies within the LOH11A region, but that its exons are not mutated in tumors. The potential for RRM1 to act as a tumor suppressor is discussed.

Relative cytotoxic activity of immunotoxins reactive with different epitopes on the extracellular domain of the c-erbB-2 (HER-2/neu) gene product p185.

Different epitopes on the extracellular domain of the HER-2 receptor can serve as distinct targets for immunotoxins. To determine the optimal epitope target for immunotoxin therapy, 7 anti-HER-2 ricin A chain murine monoclonal immunotoxins, each reactive with different epitopes of HER-2 receptor, were tested for cytotoxic activity. The immunotoxins produced 1.2-4.6 logs of cytotoxicity in limiting dilution clonogenic assays with 2 breast cancer cell lines that overexpressed HER-2. Cytotoxicity did not correlate with immunoglobulin isotype, binding affinity, relative position of epitopes or internalization of the anti-HER-2 immunotoxins. Interestingly, the most and least effective immunotoxins bound to epitopes in very close proximity. Competitive binding assays with unconjugated antibodies have previously indicated that our antibodies recognized epitopes that are arranged in a linear array. To orient this relative epitope map, deletions were prepared in the HER-2/neu gene and these mutant constructs were expressed in NIH3T3 cells. Epitope expression was determined by antibody binding and radioimmunoassay. Epitopes targeted by the PB3, 454C11 and NB3 antibodies are localized N-terminal to the epitopes recognized by ID5, BD5, 741F8 and 520C9 antibodies. The 2 non-conformational epitopes PB3 and NB3 were localized to regions corresponding to amino acides 78-242 of the p185(HER-2) protein.

A 1.4-Mb high-resolution physical map and contig of chromosome segment 11p15.5 and genes in the LOH11A metastasis suppressor region.

The centromeric part of chromosome segment 11p15.5 contains a region of frequent allele loss in many adult solid malignancies. This region, called LOH11A, is lost in 75% of lung cancers and is thought to contain a gene that may function as a metastasis suppressor. Genetic complementation studies have shown suppression of the malignant phenotype including reduction of metastasis formation. We constructed a high-resolution physical map and contig over 1.4 Mb that includes the beta-hemoglobin gene cluster and the gene for the large subunit of ribonucleotide reductase (RRM1). Through sequencing and computerized analysis, we determined that this region contains an unusually large number of transposable elements, which suggests that double-stranded DNA breaks occur frequently here. Twenty-two putative genes were identified. Because of its location at the site of maximal allele loss in the 650-kb LOH11A region and previous functional studies, RRM1 is the most likely candidate gene with metastasis suppressor function. The malignant phenotype, in this case, results from a relative loss of function rather than a complete loss.

In vitro chemosensitivity testing of human non-small cell lung cancer cell lines.

Three human non-small cell lung cancer (non-SCLC) cell lines, one each derived from squamous cell carcinoma, adenocarcinoma, and large cell carcinoma were tested for chemosensitivity to 23 cytostatic agents using the soft agarose clonogenic assay. Results were compared with in vitro responses of SCLC cell lines derived from patients with and without prior chemotherapy and with in vivo phase II clinical trials in non-SCLC. Carboplatin, daunorubicin, paclitaxel, and teniposide were active in two non-SCLC cell lines and actinomycin D, bleomycin, etoposide, irinotecan, and mithramycin were active in one cell line. The cell line from the treated SCLC patient was less sensitive to cytostatic agents than the cell line from the untreated SCLC patient and had a sensitivity pattern comparable to non-SCLC cell lines. These data are consistent with clinical experience in non-SCLC and suggest that non-SCLC cell lines in conjunction with the soft agarose clonogenic assay can be useful for in vitro drug screening. They confirm that carboplatin and paclitaxel are among the most active drugs in non-SCLC and suggest that daunorubicin and teniposide may be more active then their more frequently used analogues.

Cell surface density of p185(c-erbB-2) determines susceptibility to anti-p185(c-erbB-2)-ricin A chain (RTA) immunotoxin therapy alone and in combination with anti-p170(EGFR)-RTA in ovarian cancer cells.

Approximately 30% of ovarian and breast cancers overexpress p185(c-erbB-2) with as many as 10(6) receptors/cell. Normal cells have as few as 10(4) receptors/cell. We have examined the susceptibility of SKOv3 human ovarian cancer cells to anti-c-erbB2 antibodies and immunotoxins as a function of c-erbB-2 density on the cell surface. A panel of SKOv3 clones that expressed different densities of p185(c-erbB-2) receptor were generated through transfection with the c-erbB-2 gene. A significant correlation was found between p185(c-erbB-2) density and susceptibility to killing by anti-p185(c-erbB-2)-ricin A chain (anti-p185(c-erbB-2)-RTA) immunotoxins. With 10(5) copies/cell of p185(c-erbB-2), <10% of clonogenic ovarian cancer cells could be eliminated, whereas in clones that expressed 10(6) copies/cell of p185(c-erbB-2), 99.9% of clonogenic tumor cells were killed. In cell lines that overexpressed p185(c-erbB-2) and also expressed p170(EGFR), anti-p185(cerbB-2)-RTA and anti-p170(EGFR)-RTA immunotoxins exerted synergistic cytotoxicity. Treatment with the two immunotoxins could eliminate 99.99% of clonogenic cells. Importantly, tumor cells that had survived first treatment with anti-p185(c-erbB2)-RTA alone still retained sensitivity to repeat treatment with the same immunotoxin and also proved susceptible to the synergistic cytotoxicity of anti-p185(cerbB-2)-RTA in combination with anti-p170(EGFR)-RTA. Growth characteristics of the clones expressing various levels of p185(c-erbB-2) were also studied. No correlation was found between p185(c-erbB-2) expression levels and the rate of anchorage-dependent growth, anchorage-independent growth, or in vivo growth in nude mice.

Molecular characterization of the human delta opioid receptor in lung cancer.

A variety of neuropeptide receptors have been detected in human lung cancer. They are thought to play a role in autocrine/paracrine regulation of cell growth, and may be clinically useful as diagnostic, prognostic or therapeutic targets. The current study characterizes the molecular structure of the delta opioid receptor and its gene expression level in lung cancer cell lines relative to normal human lung using a sensitive RT-PCR approach. The goals of this investigation were a) to define the correlation between receptor binding and gene expression in lung cancer cell lines, and b) to determine the cDNA sequence integrity of this receptor in comparison to the receptor recently found in human brain. Five small cell lung cancer (SCLC) cell lines revealed size-matched RT-PCR products which strongly hybridized to the human brain delta opioid receptor probe. One of three non-small cell lung cancer (NSCLC) cell lines (NCI-H23), known to be negative by binding analysis, demonstrated low level expression. No gene expression was found in normal human lung. RT-PCR products from two SCLC cell lines (SCLC-22H and 16HC) as well as the low level expressing NSCLC cell line (NCI-23) were subjected to bidirectional DNA sequence analysis and the receptor ends were resolved using a 3'-end RACE and 5'-end gene-specific approach. The isolated cDNA sequences proved to be identical to the published human brain delta opioid receptor sequence. These data show that lung cancers with neuroendocrine features express human brain delta opioid receptors in contrast to normal lung, and that the delta opioid receptor mRNA in lung cancer is not mutated. This unique feature of lung cancer may be exploitable for diagnostic, prognostic, and therapeutic strategies.

Association of chromosome 11 locus D11S12 with histology, stage, and metastases in lung cancer.

We have reported frequent allele loss for the marker D11S12 on chromosome band 11p15.5 in human lung cancer. The smallest common region of allele loss has been refined to approximately 500 kb and is confined between D11S1758 and D11S860. Here, we investigated the association of D11S12 allele loss with epidemiologic, pathologic, and clinical parameters. Analysis of allele loss was performed by Southern blotting on a cohort of 156 patients with lung cancer, and data were interpreted with the use of a phosphorimager. Results were statistically compared with retrospectively collected variables. D11S12 allele loss was found in 88% of small cell carcinomas, 57% of squamous cell carcinomas, and 40% of adenocarcinomas. Allele loss was associated with tumor stage (p = 0.04) and was more frequent in tumors that had already metastasized. These results suggest that a gene in the D11S12 region may be responsible for the metastatic potential of lung cancer. The functional status of this gene may thus be of future value in guiding clinicians on decisions regarding adjuvant and neoadjuvant therapies for patients with lung cancer.

Growth inhibition of a human lung adenocarcinoma cell line by genetic complementation with chromosome 11.

Two regions on chromosome segment 11p15.5 have frequent allele loss in lung cancer. LOH11A is centromeric between loci D11S1758 and D11S12, and LOH11B is telomeric between HRAS and D11S1363. We studied the biological significance of this allele loss using microcell-mediated transfer of human chromosomes 11, 11p, and two radiation-reduced fragments of 11p into human lung adenocarcinoma cell lines. Chromosome 12, which has not been implicated in lung carcinogenesis, was used as a control. All four chromosome 11-containing hybrid clones showed significantly reduced tumorigenicity in nude mice and growth in liquid culture. These findings support the notion of a tumor suppressor gene located in the LOH11A region on chromosome segment 11p15.5.

Smoking, gender, and survival association with allele loss for the LOH11B lung cancer region on chromosome 11.

We have reported frequent allele loss for the marker HRAS on chromosome 11p in human lung cancer and defined the smallest common region of deletion (designated LOH11B) to approximately 500 kb. Here, we investigated the association of allele loss for LOH11B with epidemiological, pathological, and clinical parameters. Analysis of allele loss was performed using Southern blotting on a cohort of 200 patients with lung cancer, and data were interpreted with the use of a phosphorimager. Results were statistically compared with retrospectively collected variables. LOH11B allele loss was significantly associated with cigarette consumption (P = 0.009), gender (P = 0.02), and survival (P = 0.04). None of the nonsmokers had allele loss as compared with 28% of the patients with low and 43% with high cigarette consumption. Allele loss was more frequent in men (43%) than in women (11%). The median survival of patients without allele loss was 42 months compared with 25 months for patients with allele loss. These results suggest that the LOH11B region contains a gene responsible for a more malignant phenotype independent of the metastatic potential of lung cancer. They also suggest that alterations in this gene are associated with cigarette consumption and are more frequent in men than in women.

Delineation of the centromeric and telomeric chromosome segment 11p15.5 lung cancer suppressor regions LOH11A and LOH11B.

We have reported frequent allele loss for two separate regions identified by the markers D11S12 and HRAS on chromosome 11p15.5. D11S12 allele loss was associated with tumor stage and type and HRAS allele loss with cigarette consumption, sex, and survival. To positionally clone the putative tumor suppressor genes located in these regions, we here report a reduction in the size of these intervals to approximately 250 kb. The markers used, ordered from centromere to telomere, were D11S932 -D11S1331-D11S1760-D11S1323-D11S4891 (HBB)-D11S1758-D11S12-D11S988-D11S860-D11S131 8-TH-HRAS-D11S1363-D11S2071. We analyzed 44 tissue pairs from patients with primary lung cancer. The smallest common regions of allele loss were located between D11S1758 and D11S12 in the centromeric region of chromosome segment 11p15.5 (region of LOH on chromosome 11 in lung cancer, LOH11A) and between HRAS and D11S1363 in the telomeric region (region of LOH on chromosome 11 in lung cancer, LOH11B). Loss of heterozygosity was observed in 24/39 (62%) primary lung cancers informative for LOH11A and in 17/34 (50%) informative for LOH11B. The pattern of allele loss strongly suggests that two lung cancer suppressor genes are located on chromosome segment 11p15.5, one between D11S1758 and D11S12 and the other between HRAS and D11S1363. The estimated physical size of each of these regions is 250 kb.

Heregulin and agonistic anti-p185(c-erbB2) antibodies inhibit proliferation but increase invasiveness of breast cancer cells that overexpress p185(c-erbB2): increased invasiveness may contribute to poor prognosis.

Overexpression of p185(c-erbB2) (p185/NEU/HER2) by tumor cells is associated with a poor prognosis in many but not all studies of breast and ovarian cancer. The poor prognosis associated with overexpression of p185(c-erbB2) could result from an increased growth rate or increased invasive potential. The p185(c-erbB2) tyrosine kinase receptor can be activated with agonistic antibodies directed against p185(c-erbB2) or with the ligand heregulin through a combinatorial interaction with erbB3 or erbB4. Consequently, we have asked whether heregulin or agonistic antibodies increase anchorage-independent growth or invasiveness of the SKBr3 breast cancer cell line, which overexpresses p185(c-erbB2). Incubation of SKBr3 breast cancer cells with heregulin inhibited anchorage-independent growth while enhancing tyrosine phosphorylation of p185(c-erbB2). Heregulin treatment also increased adhesion of SKBr3 cells to plastic and increased invasiveness of tumor cells into Matrigel membranes while increasing expression of the CD44 (HCAM) and CD54 (ICAM-1) adhesion molecules. Tumor cell invasion of Matrigel membranes was partially blocked by either anti-CD44 or anti-CD54 antibodies, indicating a role for these adhesion molecules in the invasion process. Compatible with the increased invasiveness, heregulin increased expression of the matrix metalloproteinase 9. In contrast, the agonistic anti-p185(c-erbB2) antibody ID5 induced only a subset of the responses induced by heregulin. ID5 induced tyrosine phosphorylation of p185(c-erbB2), increased invasiveness, and increased expression of CD44. Despite the similarity of effects of ID5 and heregulin on some outcomes, the ID5 antibody failed to increase adhesion to plastic, expression of CD54, or production of matrix metalloproteinase 9. Thus, the ID5 agonistic anti-p185(c-erbB2) antibody mimics rather than antagonizes some but not all of the actions of heregulin. Moreover, the poor prognosis of breast and ovarian cancers that overexpress p185(c-erbB2) could relate in part to enhanced invasiveness rather than to increased proliferative capacity.

Combinations of multiple serum markers are superior to individual assays for discriminating malignant from benign pelvic masses.

To determine whether measurement of the levels of multiple tumor markers in the preoperative serum of women presenting with a pelvic mass distinguished benign from malignant disease better than the assay of CA 125 alone, sera from 429 patients, 192 of whom had malignant histology, were assayed for 8 different markers: CA 125, macrophage colony-stimulating factor, OVX1, lipid-associated sialic acid (LASA), CA15-3, CA72-4, CA19-9, and CA54/61. The sensitivity and specificity of CA 125 alone (> 35 U/ml) was 78.1 and 76.8%, respectively. A panel consisting of CA 125, OVX1, LASA, CA15-3, and CA72-4 had a sensitivity of 83.3% and specificity of 84.0% when two or more markers were elevated. Using the concentrations of these five markers, logistic regression analysis had a sensitivity of 85.4% and a specificity of 83.1%. Considering the values of markers in different sequences, classification and regression tree analysis substantially improved the sensitivity to 90.6% and the specificity to 93.2%. When applied in clinical practice this approach could improve the management of women presenting with a pelvic mass and may also have application in screening for ovarian cancer.

The tyrosine kinase activity of the C-erbB-2 gene product (p185) is required for growth inhibition by anti-p185 antibodies but not for the cytotoxicity of an anti-p185-ricin-A chain immunotoxin.

Our previous studies have demonstrated that 7 of 10 IgG antibodies against distinct epitopes on the extracellular domain of the c-erbB-2 gene product (p185) inhibit the anchorage-independent growth of SKBr3 human breast-cancer cells that overexpress this transmembrane tyrosine kinase growth-factor receptor. Two of 7 growth-inhibitory antibodies also block the binding and function of the gp30 and p75 c-erbB-2 ligands. In this report we have studied phosphorylation of p185 and different intracellular substrates after binding of antibodies that do or do not inhibit tumor-cell growth. A correlation has been found between antibodies that inhibit growth and the intensity of tyrosine phosphorylation of p185. At late intervals, serine phosphorylation of at least 3 intracellular substrates is increased preferentially by growth-inhibitory antibodies. To test the importance of p185 kinase activity more critically, NIH3T3 cells were transfected with an expression vector containing the full-length human c-erbB-2 gene (cell line 17313), c-erbB-2 with deletion of the kinase region from codons 751-979 (cell line 9309) or c-erbB-2 with deletion of most of the intracellular domain from codons 684-1255 (cell line 9310). Unconjugated antibodies inhibited anchorage-independent growth of 17313 cells as well as SKBr3 cells, but did not inhibit growth of either 9309 or 9310 cells. In contrast, the cytotoxic effect of anti-p185-ricin A chain (RTA) conjugates was comparable for 17313, 9309 and 9310. The tyrosine-kinase activity of p185 is required for growth inhibition mediated by unconjugated anti-p185 antibodies, but not for the cytotoxic activity of anti-p185-RTA immunotoxins.