Proteins binding to cisplatin-damaged DNA in human cell lines.

Cisplatin (CDDP) is a highly effective, frequently used cancer chemotherapeutic drug employed in the treatment of several human malignancies including ovarian, testicular, and bladder cancers. A common problem encountered with cisplatin therapy is intrinsic or acquired resistance to this drug. While the mechanisms of resistance to cisplatin, and other chemotherapeutic agents, are not fully understood, one factor affecting the cellular response to CDDP may result from differences in the level of specific proteins that recognize CDDP-damaged DNA. We have developed a damaged DNA affinity precipitation technique that allows the direct visualization and characterization of cellular proteins that bind to cisplatin-damaged DNA. In the present study we have utilized this method to analyze proteins present in several mammalian cell lines that bind to cisplatin-damaged DNA. We demonstrate that HeLa cells, resistant to CDDP cytotoxicity, contain high levels of high-mobility-group proteins 1 and 2, which bind to CDDP-DNA. We also show that xeroderma pigmentosum cells of different genetic complementation groups contain variable levels of a 45-kDa protein that binds to CDDP-DNA. Thus, our results indicate that different human cell lines demonstrate qualitative and quantitative differences in the expression of cisplatin-damaged DNA binding proteins.

Localization of the binding region of high mobility group protein 2 to cisplatin-damaged DNA.

Cisplatin (CDDP) is an effective cancer chemotherapeutic drug used in the treatment of several human malignancies. The effectiveness of cisplatin therapy is limited by intrinsic resistance of tumors to this drug as well as the development of secondary tumors, which are also drug resistant. A potential mechanism influencing the sensitivity of cells to CDDP may result from the interaction of specific proteins with CDDP-damaged DNA (CDDP-DNA). In an earlier report, we demonstrated that high mobility group (HMG) proteins 1 and 2 bind with high affinity to CDDP-DNA. In the present study partial proteolytic digestion was used to localize the binding region of HMG2. A proteolytic fragment of approximately 20 kDa, containing the amino-terminal region of the protein, maintains the ability to bind with high affinity to CDDP-DNA, while an amino-terminal fragment of 14 kDa binds with slightly reduced affinity. In contrast, a peptide fragment lacking 51 NH2-terminal amino acids from HMG2 has greatly reduced affinity for damaged DNA. Recombinant peptide fragments containing HMG box 1 or HMG box 2 bind weakly to damaged DNA, while a recombinant fragment containing HMG boxes 1 and 2 binds with high affinity. Hence, our results indicate that the amino-terminal region of HMG2 contains the damaged DNA binding recognition site and that both HMG boxes 1 and 2, present in the parental molecule, are required for high affinity binding of this protein to CDDP-DNA.

Characterization of high mobility group protein binding to cisplatin-damaged DNA.

cis-Diamminedichloroplatinum (II) (cisplatin, CDDP) is a widely used chemotherapeutic agent. While many tumors are highly responsive to CDDP, certain tumors are resistant to this drug, limiting its efficacy. The anti-tumor activity of CDDP is believed to result from its coordination bonding to chromosomal DNA. Alterations in tumor cell sensitivity to CDDP may result from the presence or absence of protein(s) which specifically recognize CDDP-damaged DNA. We have developed a damaged-DNA affinity precipitation assay that allows the direct identification of cellular proteins that bind to CDDP-damaged DNA. Using this procedure, we have identified several proteins which specifically bind to CDDP-damaged DNA. Two of these proteins have been identified as high mobility group proteins (HMG) 1 and 2 in the current report, we have characterized the binding of these proteins to CDDP-DNA. The calculated Kd of binding to CDDP-damaged DNA was 3.27 x 10(-10) for HMG1 and 1.87 x 10(-10) for HMG2. Using highly specific chemical modifying reagents, we have determined that Cys residues play an important role in protein binding. We also observed that HMG2 will bind to DNA modified with carboplatin and iproplatin although to a lesser extent than to DNA damaged with CDDP. Thus, our results indicate that HMG 2 binds with high affinity to DNA modified with therapeutically active platinum compounds. In addition, our findings suggest that thiol groups play an essential role in the binding of HMG1 and HMG2 to CDDP-DNA.

Clinical stage II non-small cell lung cancer treated with radiation therapy alone. The significance of clinically staged ipsilateral hilar adenopathy (N1 disease).

BACKGROUND: The prognosis of patients with clinically staged hilar nodal involvement (Stage N1) or clinical Stage II non-small cell lung cancer (NSCLC, Stage T1-2N1M0) treated with radiation therapy (RT) alone is not well established. METHODS: Records of 758 patients with clinical Stage I-III NSCLC treated with RT were reviewed. Sixty-two patients were identified with clinical Stage II NSCLC, and 126 patients had Stage N1 disease. RESULTS: The median survival time (MST) of the 62 patients with clinical Stage II disease was 17.9 months, with 1-year, 2-year, 3-year, and 5-year overall actuarial survival rates of 70%, 33%, 20%, and 12%, respectively. The survival of patients with clinical Stage II disease was significantly better than that of 389 patients with clinical Stage IIIA disease (MST, 11.3 months; P < 0.008) and 267 patients with clinical Stage IIIB disease (MST, 9.8 months; P = 0.0003), but it was similar to that of 40 patients with clinical Stage I lesions (MST, 15.0 months). Patients with performance statuses of 0-1 lived longer than those with a status of 2 or more (MST, 22.8 versus 6.1 months; P < 0.0001). The median survival for patients with N0, N1, N2, and N3 disease was 13.7, 12.6, 10.9, and 9.1 months, respectively. Patients with Stage N0-1 disease (MST, 13.2 months) had significantly improved MST compared with those with Stage N2-3 disease (MST, 10.3 months). CONCLUSIONS: The survival of patients with clinical Stage II NSCLC treated with RT alone was significantly better than that of those with clinical Stage IIIA or IIIB disease. It was comparable to that of patients with clinical Stage I lesions. The clinical staging of nodal involvement limited to the ipsilateral hilum does not necessarily portend a worse prognosis than that of patients with clinical Stage N0 disease. The absence of clinically evident Stage N2-3 disease is of significant predictive value for patients with NSCLC treated with RT.

Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2.

The biochemical processes responsible for the recognition and repair of cisplatin-damaged DNA in human cells are not well understood. We have developed a damaged DNA affinity precipitation technique that allows the direct visualization and characterization of cellular proteins that bind to cisplatin-damaged DNA. The method separates damaged DNA-binding proteins from complex radiolabeled cell mixtures and further resolves them into individual polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique is complementary to gel retardation and Southwestern blotting analyses that have been previously used to identify cellular components that specifically bind to cisplatin-damaged DNA. Using this technique, we have characterized a set of HeLaS3 nuclear proteins of 26.5, 28, 90, and 97 kDa that specifically bind to cisplatin-DNA adducts. Competition studies with soluble cisplatin-damaged DNA confirmed these findings. The major cisplatin-damaged DNA-binding proteins of 26.5 and 28 kDa recognized adducts of DNA modified with cisplatin but not with its trans-isomer or with UV radiation. These proteins were purified 450-fold to near homogeneity by ion-exchange and cisplatin-damaged DNA affinity chromatography. Amino-terminal sequence analysis showed that the 26.5- and 28-kDa proteins were identical to high mobility group (HMG) proteins HMG-2 and HMG-1, respectively.

Comparison of outcome between clinically staged, unresected superior sulcus tumors and other stage III non-small cell lung carcinomas treated with radiation therapy alone.

Several studies suggest that patients with non-small cell lung carcinoma (NSCLC) of the superior sulcus fare better after radiation therapy than those patients with comparable tumors at other thoracic sites. There is limited data on stage-by-stage comparisons between patients with superior sulcus tumors (SST) and non-SST (NSST). Thirty patients had SST among 656 patients with American Joint Committee on Cancer clinically staged IIIA (n = 389) and IIB (n = 267) primary NSCLC who received definitive once-daily radiation therapy. The median patient age, sex ratio, histologic findings, grade, weight loss, and performance status were similar for SST and NSST. Minimum follow-up was 24 months, with 88% of patients followed until death. The survival of patients with SST (median, 10.3 months) was similar to that of patients with tumors at other pulmonary sites (median, 10.8 months; P = 0.39). Survival for favorable patients with performance status 0 to 1 and weight loss of 5% or less was comparable between patients with SST (median, 15.0 months) and NSST were similar for patients with SST and NSST (P = 0.48). The brain was the site of first failure in 20% of patients with SST and 10% of patients with NSCLC at other sites (P = 0.10). The lack of apparent difference in outcome of comparably staged patients with SST and NSST treated with radiation alone may have significant therapeutic implications.

Adverse influence of younger age on outcome in patients with non-small cell lung carcinoma (NSCLC) treated with radiation therapy (RT) alone.

Treatment outcome of 63 patients younger than 50 years of age initiated on a course of once-daily definitive radiation therapy without concurrent or preirradiation chemotherapy for clinical Stages I-III unresected non-small cell lung carcinoma from 1978 to 1988 was compared to the outcome of 695 patients over the age of 50. Follow-up ranged from 24-110 months with follow-up until death in 88% of patients. The actuarial overall survival rate for all patients was 22% at 2 years with a median survival time of 11.5 months. Patients less than 50 and greater than or equal to 50 years old were similar in male:female ratio, distribution of histologic subtype, performance status, and extent of weight loss. Poorly differentiated histologic grade was more prevalent among the younger patients (59% vs 41%, p = .005). Ninety-four percent of younger patients and 86% of older patients had clinical stage III disease (p = NS). Survival was significantly worse for patients who were younger than 50 years old (p = .05), with a median survival time of 7.8 months. Median survival time for those patients 50 years of age or older was 12.4 months. Poorer survival outcome among young patients was most pronounced among patients with unfavorable characteristics of poor performance status (greater than or equal to 2) or weight loss (greater than 5%) (p = .002). Distant failure (p = .029) and brain failure (p = .003) as initial site of relapse was more common among younger patients. Among young patients, poor histologic grade was associated with both distant failure (p = .003) and brain metastasis (p = .002). The difference in distribution of histologic grade, incidence of distant failure, particularly in the brain, and poorer survival outcome among patients less than 50 may be indicative of more aggressive tumor behavior in the younger patients. These results indicate that patients less than 50 may require alternate treatment strategies. Age should be considered a stratification variable in non-operative randomized trials of non-small cell lung carcinoma which include patients with non-favorable characteristics.

Effect of caffeine on the expression of a major X-ray induced protein in human tumor cells.

We have examined the effect of caffeine on the concomitant processes of the repair of potentially lethal damage (PLD) and the synthesis of X-ray-induced proteins in the human malignant melanoma cell line, Ul-Mel. Caffeine administered at a dose of 5mM after X radiation not only inhibited PLD repair but also markedly reduced the level of XIP269, a major X-ray-induced protein whose expression has been shown to correlate with the capacity to repair PLD. The expression of the vast majority of other cellular proteins, including seven other X-ray-induced proteins, remained unchanged following caffeine treatment. A possible role for XIP269 in cell cycle delay following DNA damage by X irradiation is discussed.

Mr 92,000 gelatinase release correlates with the metastatic phenotype in transformed rat embryo cells.

In a previous study we described a correlation between the metastatic potential of transformed rat embryo cells (REC) and their release of type IV collagenolytic activity (S. Garbisa et al., Cancer Res., 47: 1523-1528, 1987). In the present study, we have identified a Mr 92,000 gelatinase released exclusively by metastatic cell lines in vitro; seven of eight highly metastatic REC lines transformed by H-ras or H-ras plus v-myc released this enzyme. In contrast, the Mr 92,000 gelatinase was not detected in two separate nontumorigenic REC lines immortalized with H-ras or c-myc or in four independent tumorigenic REC lines (transformed by H-ras plus adenovirus E1A) with markedly reduced metastatic potential. Study of the Mr 92,000 gelatinase in tumor explants showed that its expression may be modulated in vivo. Not only was Mr 92,000 release enhanced in tumor explants from cell lines which released it in vitro, but its expression was evident in three primary tumors and six metastatic tumors from the one metastatic cell line that failed to release it in vitro. Explants from the nonmetastatic cell lines grown as tumors showed no Mr 92,000 gelatinase release. The heterogeneous expression of a number of other gelatinases with molecular weights of 52,000, 56,000, 62,000, 68,000, 80,000, and 240,000 was observed, but no correlation with metastatic potential was apparent. The Mr 92,000 gelatinase had the characteristics of a secreted neutral metalloproteinase. It may be responsible for the type IV collagenolytic activity reported previously in conditioned medium from metastatic transformed REC and could in part be responsible for the differences in metastatic potential in these cell lines.

Identification and characterization of X-ray-induced proteins in human cells.

In order to investigate the biochemical events involved in potentially lethal DNA damage repair (PLDR), we have identified a pleiotropic protein expression response that is activated upon X-irradiation of confluence-arrested human malignant melanoma (U1-Mel) cells. Plateau-phase U1-Mel cells were selected because of their extraordinary capacity for PLDR. Eight major X-ray-induced polypeptides (XIPs) of Mr 126,000-275,000 (i.e., XIP126 through XIP275) were detected by resolving L-[35S]methionine-labeled whole cell extracts using two-dimensional gel electrophoresis. XIPs were found in unirradiated, proliferating U1-Mel cells, shut off under plateau-phase conditions and resynthesized in response to X-irradiation. The expression of three classes of proteins was affected by X-irradiation. Class I proteins, XIP145 and XIP269, were induced linearly with increasing X-ray doses. The rate of synthesis of class II proteins, XIP126, XIP135, XIP138, XIP141, XIP147, and XIP275, increased linearly with low X-irradiation doses, but plateaued at doses of 150-250 cGy. In contrast, the expression of class III proteins, 47,000 and 254,000 Mr proteins, decreased with increasing X-ray doses. Tumor, cancer-prone, and normal human cells, which represent a wide range of cells with varied repair capacities, were investigated to better understand the role of XIPs in DNA damage responses. X-irradiated normal and tumor cells induced the synthesis of XIP145 and XIP269. A strong correlation between the induction of XIP269 and PLDR capacity, as measured by delayed plating of plateau-phase cells, was noted. XIP269 was present in six of seven normal and tumor cells types, but was completely absent in cells from patients with Bloom's syndrome and ataxia telangiectasia. X-irradiated Fanconi's anemia and xeroderma pigmentosum cells synthesized low levels of XIP269. The majority of XIPs synthesized by X-irradiated cells from cancer-prone patients were of low molecular weights. A number of XIP expression characteristics suggest their role in either gross chromosomal PLDR and/or in X-ray adaptivity responses: (a) XIP expression was inhibited by 1 microgram/ml cycloheximide, a dose which decreased survival 6-fold during PLDR holding and resulted in greater than 80% inhibition of protein synthesis; (b) XIP expression was specific for ionizing radiation damage, since heat shock, hypoxia, and alkylating agents failed to induce their synthesis; (c) the time course of expression was long, with the first appearance of XIPs at 3 h and maximal expression at 4 h.

Medulloblastoma at the joint center for radiation therapy between 1968 and 1984. The influence of radiation dose on the patterns of failure and survival.

In order to assess the efficacy of high-dose irradiation to the posterior fossa and low-dose irradiation to the spinal axis, we reviewed the results of 60 patients with biopsy-proven medulloblastoma treated at the Joint Center for Radiation Therapy (JCRT) between 1968 and 1984. The 5- and 10-year actuarial survival rates for all patients were 68% and 44%, respectively. The median time to recurrence was 19 months. Extent of surgery, age, and radiation dose to the posterior fossa all were of prognostic value. Complete or subtotal gross resection appeared to be a favorable prognostic indicator compared with biopsy only (P less than 0.05), with a 69% versus 40% actuarial survival rate at 5 years, respectively. Infants 2 years of age or less had a diminished 5-year actuarial survival rate of 48% (P less than 0.05) compared with older age groups. The posterior fossa was the predominant site of recurrence and accounted for 78% of all failures. Local control in the posterior fossa was dose dependent. Seventy-nine percent of the tumors that received 5000 cGy or greater were controlled versus only 33% of the tumors that received less than 5000 cGy (P less than 0.02). There were no supratentorial failures, and there was only one isolated spinal cord failure. There were no solitary spinal failures in 24 patients who received a median dose of only 2400 cGy to the spinal axis. We concluded that low-dose irradiation to the spine and whole brain may be indicated with maintenance of a posterior fossa dose of greater than 5000 cGy.

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells.

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.

Coprecipitation of heat shock proteins with a cell surface glycoprotein.

Monoclonal antibodies recognizing a mouse cell surface glycoprotein of Mr 90,000 were found to coprecipitate the Mr 70,000 and 72,000 heat shock-induced proteins of NIH/3T3 cells. These two smaller proteins were among the most abundant components of heat-treated NIH/3T3 cells. The Mr 70,000 component was not detected in normal cells whereas there was a low rate of incorporation of [35S]methionine into the Mr 72,000 polypeptide in the absence of heat shock. Tryptic peptide mapping and two-dimensional gel electrophoresis indicated that the coprecipitated and heat shock-induced polypeptides were identical and that the Mr 70,000 and 72,000 components contained homologous peptides. Also, the heat shock proteins had extensive structural homology with a cytoskeleton-associated protein of HeLa cells. The results suggest that the Mr 90,000 cell surface glycoprotein and the Mr 70,000 and 72,000 heat shock-inducible proteins mediate an association between the plasma membrane and the cell cytoskeleton.

Murine cell surface glycoproteins. Identification, purification, and characterization of a major glycosylated component of 110,000 daltons by use of a monoclonal antibody.

A 110,000-dalton plasma membrane glycoprotein of mouse cells has been identified, purified, and characterized by use of a xenogeneic monoclonal antibody. The glycoprotein was a major component of the NIH/3T3 cell surface. It contained 15% of [3H]glucosamine incorporated into cell proteins and was composed of at least 16 isomorphic variants with apparent molecular weights from 100,000 to 120,000 and isoelectric points between 6.5 and 8.1. The glycoprotein constituted 0.1% of total cell protein, as measured by the yield of purified protein, and there were over 10(6) antibody-binding sites/cell. Immunoprecipitation from pulse-chase labeled cells showed that the 110,000-dalton glycoprotein was initially synthesized as a 92,000-dalton microsomal polypeptide which gradually was converted to the mature surface form. The turnover time of the surface form was at least 20 h. The antigenic determinant recognized by the monoclonal antibody was species-specific and nonpolymorphic. It was present in high concentration on most dividing murine cells in culture, whereas the concentration in normal mouse tissues differed widely. Among lymphoid tissues, antigen concentration was enriched in bone marrow as compared to spleen and thymus. Among nonlymphoid tissues, antigen concentration was 10-fold greater in kidney than in brain, liver, and skeletal muscle. The glycoprotein was purified without loss of antigenic activity by antibody affinity chromatography. The single step procedure yielded 3 mg of pure glycoprotein from 3 g of crude cell extract. The purification and characterization of this major membrane protein provide a basis for further study of its cell surface structure and function.

Gene for a major cell surface glycoprotein of mouse macrophages and other phagocytic cells is on chromosome 2.

A gene controlling the expression of a polymorphic 92,000-dalton glycoprotein of mouse macrophages and granulocytes has been identified. This glycoprotein was previously shown to be the major iodinated, trypsin-sensitive component of the murine phagocyte cell surface. The gene has been provisionally designated Pgp-1 for phagocyte glycoprotein 1. Expression of the glycoprotein was measured by monoclonal antibody binding to a polymorphic antigenic determinant. Antibody binding to cells of positive strains of mice was proportional to cell number, whereas binding to cells of negative strains was insignificant. The concentration of the antigen in cells of heterozygous mice was approximately 50% of that in homozygous mice. Thirteen of 33 recombinant inbred strains of mice were positive, with binding values 100-fold over background, suggesting that a single gene controlled expression of the antigen. Segregation of the antigen correlated with markers on chromosome 2. The segregation of Pgp-1, with nonagouti coat color (a) and hemolytic complement (Hc) activity among progeny of (C3H/HeJ x DBA/2J)F1 x DBA/2J mice confirmed the single gene control and the chromosomal assignment. Another gene on chromosome 2, Ly-m11, was also typed by using (BALB/cJ x C57BL/6J)F1 x C57BL/6J mice. The data from both of these crosses indicated the following gene order: Hc--Pgp-1--Ly-m11--a.

Murine cell surface glycoproteins. Characterization of a major component of 80,000 daltons as a polymorphic differentiation antigen of mesenchymal cells.

Monoclonal antibodies reactive with NIH/3T3 cell surface antigens were obtained from hybridomas of murine myeloma cells fused to spleen cells of rats immunized with NIH/3T3 cell plasma membranes. Four of the antibodies, of forty that have been studied, appeared to react with allospecific antigenic determinants: they bound to NIH/3T3 cells but not to BALB/ 3T3 cells. Each of these four antibodies immunoprecipitated a glycoprotein of about 80,000 daltons that migrated to an isoelectric point of about pH 5.0. Polypeptides of identical molecular weight and isoelectric points, and yielding the same proteolytic cleavage fragments, were present in BALB/3T3 cells, but were not antigenically reactive. The 80,000-dalton glycoprotein was a major constituent of the plasma membrane. It was a predominant lactoperoxidase iodinated component of intact NIH/3T3 cells, and saturation binding of 125I-labeled antibody indicated that there were about 10(6) antigenic sites/cell. Studies of the distribution of the immunoreactive glycoprotein among different strains of mice confirmed the polymorphic expression of the determinant: Spleen cells of BALB/c, DBA/1, DBA/2, and CBA mice did not bind anti-80,000-dalton glycoprotein monoclonal antibodies, whereas spleen cells of a large number of other strains of mice were positive for antibody-binding. The antigenic reactivity varied markedly among different cell lines and was greatest with the NIH/3T3 mouse embryo fibroblast, G8-1 Swiss Webster myoblast, and IC-21 SV40-transformed C57BL/6 mouse peritoneal macrophage. The properties of the 80,000-dalton glycoprotein characterized this molecule as a new cell surface differentiation alloantigen of murine mesenchymal cells.