Frequency of homozygous deletion at p16/CDKN2 in primary human tumours.

Many tumour types have been reported to have deletion of 9p21 (refs 1-6). A candidate target suppressor gene, p16 (p16INK4a/MTS-1/CDKN2), was recently identified within the commonly deleted region in tumour cell lines. An increasing and sometimes conflicting body of data has accumulated regarding the frequency of homozygous deletion and the importance of p16 in primary tumours. We tested 545 primary tumours by microsatellite analysis with existing and newly cloned markers around the p16 locus. We have now found that small homozygous deletions represent the predominant mechanism of inactivation at 9p21 in bladder tumours and are present in other tumour types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicates a 170 kb minimal region that includes p16 and excludes p15.

Cell adhesion molecules as tumour suppressors.

Cell adhesion molecules, a diverse group of proteins expressed on the cell surface, have been implicated in numerous important cellular functions ranging from controlling morphogenesis to suppressing tumourigenesis. In this article, we discuss evidence supporting the idea that at least some proteins involved in cell adhesion may suppress tumourigenesis through influences on cell growth, differentiation and/or invasion. These studies suggest that some cell adhesion molecules may be encoded by tumour suppressor genes.

NIH3T3 cells expressing the deleted in colorectal cancer tumor suppressor gene product stimulate neurite outgrowth in rat PC12 pheochromocytoma cells.

The Deleted in Colorectal Cancer (DCC) gene is a candidate tumor suppressor gene that is predicted to encode a transmembrane polypeptide with strong similarity to the neural cell adhesion molecule (N-CAM) family. Previous studies have suggested that several different N-CAMs, when expressed in non-neuronal cell types can stimulate neurite outgrowth from PC12 rat pheochromocytoma cells. Based on the predicted structural similarity of DCC to N-CAMs, we sought to determine whether NIH3T3 cells expressing DCC could stimulate neurite outgrowth in PC12 cells. We found that NIH3T3 cell lines expressing DCC could stimulate PC12 cells to extend neurites. Supernatants from DCC-transfected NIH3T3 cells did not induce neurite outgrowth above background levels, suggesting that cell-cell interaction was required. NIH3T3 cells expressing a truncated form of DCC, lacking the majority of the cytoplasmic domain sequences, also failed to induce neurite outgrowth above the levels seen with control NIH3T3 cells, suggesting that the cytoplasmic domain of DCC was necessary for its neurite-promoting function. In contrast to NGF-mediated neurite outgrowth, the DCC-mediated response was inhibited by treatment with pertussis toxin or the combination of N- and L-type calcium channel blockers, and was unaffected by the transcriptional inhibitor cordycepin. The data suggest that the DCC protein can function in a fashion analogous to other N-CAMs to alter PC12 cell phenotype through intracellular pathways distinct from those involved in NGF signaling.

Mortality of the Monarch Butterfly (Danaus plexippus L.): Avian Predation at Five Overwintering Sites in Mexico.

Analyses of predated butterflies on the forest floor at five monarch overwintering sites in Mexico and observations of birds foraging in mixed flocks indicate that individual birds of several species have learned to penetrate the monarch's cardenolide-based chemical defense. Predation is inversely proportional to colony size and appears to be one evolutionary explanation of the dense aggregations.

Both cell proliferation and apoptosis increase with lesion grade in cervical neoplasia but do not correlate with human papillomavirus type.

Recent molecular studies suggest that the expression of high-risk but not low-risk human papillomavirus (HPV) oncoproteins E6 and E7 can significantly alter normal cell cycle regulation. The alterations in cell cycle regulation may be reflected by changes in the balance between cell growth and cell loss through apoptosis in cell populations expressing E6 and/or E7. We evaluated the kinetic indices of cell proliferation and apoptosis in a histopathological spectrum of cervical neoplasia and compared low-versus high-risk HPV-associated lesions. The cell proliferation index, as determined by detection of the nuclear antigen Ki67, increased with increasing lesion grade. Apoptotic cells were identified with terminal deoxynucleotidyl transferase-labeling of the 3'-hydroxyl ends of DNA nucleosomes. No apoptosis was observed in normal epithelium, and only occasional apoptotic cells were seen in low-grade lesions. However, there was a low but measurable apoptotic index in the higher grade lesions, which increased with lesion grade. There was no significant difference in the proliferative and apoptotic indices in similar grade lesions when stratified into low-versus high-risk HPV types. These findings suggest that apoptosis in HPV-infected lesions correlates with proliferative activity rather than HPV type.

p53 inactivation by HPV16 E6 results in increased mutagenesis in human cells.

To study the pathways associated with genomic instability in cancer, we examined UV-induced and spontaneous mutagenesis in clonal cell lines expressing human papillomavirus (HPV) proteins, either high-risk (HPV16) E6 or E7 or low-risk (HPV11) E6, in comparison to the parental RKO cells, a colon carcinoma cell line expressing only normal p53. High-risk E6 and E7 bind and functionally inactivate tumor suppressor proteins p53 and Rb, respectively, and both disrupt the G1 arrest in response to DNA damage. Low-risk HPV E6 proteins bind p53 with much lower affinity than high-risk E6 and fail to mediate p53 degradation or to disrupt the G1 checkpoint. We found that cells expressing HPV16 E6 had reduced survival and increased mutagenesis at the hprt locus when treated with low doses of UV. However, this analysis was complicated by the unexpected observation of a very high background of spontaneous mutagenesis in the unirradiated cells expressing the HPV16 E6 gene. Fluctuation analysis revealed a 5-fold elevated mutation rate in the cells expressing HPV16 E6. HPV11 E6 conferred a 2-fold elevation in the mutation rate, but HPV16 E7 had no effect. The increased spontaneous mutagenesis, therefore, appeared to be mediated by p53 inactivation and to be independent of Rb (which acts downstream of p53 in the G1 arrest pathway following DNA damage). Taken together, these findings suggest that the effect of p53 inactivation on spontaneous mutagenesis is manifested at the level of DNA repair, recombination, or coupling of transcription with one of these processes instead of by an alteration in G1 arrest.

A transforming growth factor beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability.

We have recently demonstrated that mutation of the transforming growth factor-beta (TGF-beta) receptor type II (RII) gene is characteristic of colon cancers exhibiting microsatellite instability or replication errors (RER+). Moreover, we have shown that RII mutations in these RER+ colon cancers are characteristically frameshift mutations within a 10-bp polyadenine repeat present in the RII-coding region. We now show that RII gene mutations in this polyadenine repeat are also commonly present in RER+ gastric cancers (71%). In contrast, we find these same RII gene mutations are distinctly uncommon in RER+ endometrial cancers (17%, P < 0.02). These results suggest that RII gene mutations confer a growth advantage and are selected for in RER+ cancers of both the upper and lower gastrointestinal tract. The genesis of RER+ endometrial tumors must, however, be by a different route.

Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas.

Endometrial carcinoma is the second most common tumor type in women with hereditary nonpolyposis colorectal carcinoma. Microsatellite instability (MI) has been observed in the inherited (hereditary nonpolyposis colorectal carcinoma-associated) form of endometrial carcinoma as well as in approximately 20% of presumably sporadic cases. Recent studies suggest that MI in many cell lines or xenografts derived from sporadic colorectal carcinomas is not attributable to mutations in four known human DNA mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, and hPMS2). Mutational analyses of these four MMR genes in endometrial carcinomas have not been previously reported. We analyzed nine sporadic MI-positive primary endometrial carcinomas for mutations in the above four MMR genes. Mutations were detected in two tumors (in hMSH2), and both of the mutations were acquired somatically. Immunohistochemical staining revealed a lack of expression of hMSH2 protein in the two tumors containing hMSH2 mutations. Our data suggest that mutations in these four known DNA MMR genes are not responsible for MI in the majority of sporadic endometrial carcinomas displaying this phenotype.

DPC4 gene in various tumor types.

We recently identified a novel tumor-suppressor gene, DPC4, at chromosome 18q21.1 and found that both alleles of DPC4 were inactivated in nearly one-half of the pancreatic carcinomas. Here, we analyzed 338 tumors, originating from 12 distinct anatomic sites, for alterations in the DPC4 gene. Sixty-four specimens were selected for the presence of the allelic loss of 18q and were further analyzed for DPC4 sequence alterations. An alteration of the DPC4 gene sequence was identified in one of eight breast carcinomas and one of eight ovarian carcinomas. These results indicate that whereas DPC4 inactivation is prevalent in pancreatic carcinoma (48%), it is distinctly uncommon (< 10%) in the other tumor types examined. The tissue restriction of alterations in DPC4, as in many other tumor-suppressor genes, emphasizes the complexity of rate-limiting checkpoints in human tumorigenesis.

Microsatellite instability in endometrial carcinoma.

Microsatellite instability (MI), detected as electrophoretic shifts in allele sizes of microsatellite DNA sequences, has been identified in some colorectal carcinomas. Investigators have previously attributed such microsatellite instability to replication errors (RER). The colorectal carcinomas with RER have been found to arise either sporadically or in association with the hereditary non-polyposis colorectal cancer (HNPCC) syndrome. Because endometrial carcinoma is also commonly associated with HNPCC, we studied 30 cases of endometrial carcinoma to characterize the presence of MI in these neoplasms. Seven cases (23%) showed MI. Four cases showed both Type I (large shifts) and Type II (small shifts) mutation patterns and the remaining three cases showed Type I mutations only. We conclude that MI frequently occurs in endometrial cancers and that this type of genetic alteration may be an important pathogenetic feature of this tumor type.

The DCC gene suppresses the malignant phenotype of transformed human epithelial cells.

Loss of heterozygosity and loss of expression of the deleted in colon cancer (DCC) gene is frequently observed in a number of different cancer types. To determine if the DCC gene plays a direct role in tumor suppression, wild-type full-length or truncated DCC cDNA constructs were transfected into nitrosomethylurea (NMU) transformed tumorigenic HPV-immortalized human epithelial cells that had allelic loss and reduced expression of DCC. Full-length DCC suppressed tumorigenicity whereas truncated DCC did not. Tumorigenic reversion of initially suppressed transfectants was associated with loss of DCC expression and loss or rearrangement of transfected DCC sequences. These results provide the first direct evidence that DCC is a tumor suppressor gene.

Expression of HPV16 E6 or E7 increases integration of foreign DNA.

In most invasive cervical carcinomas, high-risk human papillomavirus (HPV) DNA is integrated into the host genome, while in pre-invasive cervical lesions the viral genome is typically maintained exclusively as an episome. In contrast, integration of low-risk HPV DNA is rare, as is the association of low-risk HPVs with carcinomas. High-risk HPV integration is associated with a selective growth advantage of affected cells, and hence, integration is likely to be an important genetic alteration contributing to cervical tumor progression. Expression of high-risk, but not low-risk, HPV E6 or E7 proteins disrupts the p53-dependent G1 arrest that cells normally display in response to DNA damage. Absence of this cell cycle checkpoint may predispose cells containing high-risk HPVs to genetic instability and to the accumulation of the genetic alterations that appear to be required for HPV-associated cervical tumor progression. We hypothesized that integration of high-risk HPV DNA into the host cell genome may be facilitated by E6- and/or E7-mediated disruption of the normal DNA damage response pathway. To test this hypothesis, we assessed the integration frequency of a reporter plasmid (pHyGal) in RKO cells expressing individual E6 or E7 genes of either high-risk (HPV16) or low-risk (HPV6, HPV11) type viruses. Cells expressing HPV16 E6 or HPV16 E7 exhibited a significantly increased frequency of pHyGal integration in comparison to RKO control cells or cells expressing low-risk HPV E6 or E7. Thus, expression of high-risk, but not low-risk, E6 and E7 proteins increases the frequency of foreign DNA integration into the host genome. These findings suggest that at least some of the difference in oncogenic potential observed between high-risk and low-risk HPV types may be determined by the increased ability of high-risk HPVs to integrate into host DNA.

Loss of DCC expression in neuroblastoma is associated with disease dissemination.

DCC, a candidate tumor suppressor gene from chromosome 18q21, is most highly expressed in the developing nervous system. In vitro studies suggest a role for DCC in neuronal differentiation, and 18q allelic loss occurs in a subset of neuroblastomas. To address the hypothesis that loss of DCC function may contribute to tumorigenesis in cells of neural origin, we utilized a combination of RNase protection, immunoblotting, and immunohistochemical approaches to characterize DCC expression in 62 primary neuroblastomas and 16 neuroblastoma cell lines. The DCC protein was undetectable in 38% of the primary tumors and 56% of the cell lines. Of note, primary tumors lacking DCC expression were more likely to have been obtained from patients with disseminated or stage D disease (P = 0.01). In addition, loss of DCC expression was observed in three of six primary tumors from stage DS patients. No consistent relationship between the loss of DCC expression and N-myc amplification was observed in our studies. Our findings suggest that loss of DCC expression may contribute to the dissemination of neuroblastoma cells, perhaps through alterations in growth and differentiation pathways distinct from those regulated by N-myc.

Human papillomavirus E6 and E7 oncoproteins alter cell cycle progression but not radiosensitivity of carcinoma cells treated with low-dose-rate radiation.

PURPOSE: Low-dose-rate radiation therapy has been widely used in the treatment of urogenital malignancies. When continuously exposed to low-dose-rate ionizing radiation, target cancer cells typically exhibit abnormalities in replicative cell-cycle progression. Cancer cells that arrest in the G2 phase of the cell cycle when irradiated may become exquisitely sensitive to killing by further low-dose-rate radiation treatment. Oncogenic human papillomaviruses (HPVs), which play a major role in the pathogenesis of uterine cervix cancers and other urogenital cancers, encode E6 and E7 transforming proteins known to abrogate a p53-dependent G1 cell-cycle checkpoint activated by conventional acute-dose radiation exposure. This study examined whether expression of HPV E6 and E7 oncoproteins by cancer cells alters the cell-cycle redistribution patterns accompanying low-dose-rate radiation treatment, and whether such alterations in cell-cycle redistribution affect cancer cell killing. METHODS AND MATERIALS: RKO carcinoma cells, which contain wild-type P53 alleles, and RKO cell sublines genetically engineered to express HPV E6 and E7 oncoproteins, were treated with low-dose-rate (0.25-Gy/h) radiation and then assessed for p53 and p21WAF1/CIP1 polypeptide induction by immunoblot analysis, for cell-cycle redistribution by flow cytometry, and for cytotoxicity by clonogenic survival assay. RESULTS: Low-dose-rate radiation of RKO carcinoma cells triggered p53 polypeptide elevations, p21WAF1/CIP1 induction, and arrest in the G1 and G2 phases of the cell cycle. In contrast, RKO cells expressing E6 and E7 transforming proteins from high-risk oncogenic HPVs (HPV 16) arrested in G2, but failed to arrest in G1, when treated with low-dose-rate ionizing radiation. Abrogation of the G1 cell-cycle checkpoint activated by low-dose-rate radiation exposure appeared to be a characteristic feature of transforming proteins from high-risk oncogenic HPVs: RKO cells expressing E6 from a low-risk nononcogenic HPV (HPV 11) exposed to low-dose-rate radiation arrested in both G1 and G2. Surprisingly, despite differences in cell-cycle redistribution accompanying low-dose-rate radiation treatment associated with high-risk HPV transforming protein expression, no consistent differences in clonogenic survival following low-dose-rate radiation treatment were found for RKO cell sublines expressing high-risk HPV oncoproteins and arresting only in G2 during low-dose-rate radiation exposure vs. RKO cell sublines exhibiting both G1 and G2 cell-cycle arrest when irradiated. CONCLUSION: The results of this study demonstrate that neither HPV oncoprotein expression nor loss of the radiation-activated G1 cell-cycle checkpoint alter the sensitivity of RKO carcinoma cell lines to low-dose-rate radiation exposure in vitro. Perhaps for urogenital malignancies associated with oncogenic HPVs in vivo, HPV oncoprotein-mediated abrogation of the G1 cell-cycle checkpoint may not limit the potential efficacy of low-dose-rate radiation therapy.

Use of keratin 903 as an adjunct in the diagnosis of prostate carcinoma.

The identification of basal cells is often helpful in excluding a diagnosis of prostate carcinoma. However, it can be difficult to distinguish basal cells from underlying fibroblasts or an artifactual two-cell layer in neoplastic glands. To determine the usefulness of anti-keratin antibody 903 for identifying basal cells in glandular patterns sometimes confused with carcinoma, we examined frozen sections from radical prostatectomy specimens and formalin-fixed needle biopsy, radical prostatectomy, and transurethral resection specimens. Atrophic glands, basal cell hyperplasia, intraductal severe dysplasia and various grades of carcinoma were examined. Also evaluated were cases of atypical adenosis, defined as clusters of small glands that mimic low-grade carcinoma yet focally appear to have a basal cell layer and merge with more recognizable benign glands. Almost all normal glands showed some staining, although it was often discontinuous with formalin fixation. Intraductal dysplasia stained in a manner similar to normal glands. Ninety-two percent of atrophic glands and 88% of glands in basal cell hyperplasia stained. Sixty-one percent of the glands in atypical adenosis stained intensely but discontinuously. All grades of adenocarcinoma lacked any immunoreactivity. These results indicate that keratin 903 is useful in the diagnosis of prostatic carcinoma because positive staining identifies a questionable focus as benign whereas negative staining helps to substantiate the diagnosis of carcinoma.

Reproducibility of the diagnosis of endometrial hyperplasia, atypical hyperplasia, and well-differentiated carcinoma.

Many studies have attempted to identify histologic features that aid in the distinction of atypical hyperplasia (AH) from hyperplasia without atypia and well-differentiated endometrioid carcinoma, but few have evaluated the reproducibility of these diagnoses. Five pathologists independently reviewed 100 endometrial biopsy and curettage specimens chosen to represent the entire spectrum of proliferative lesions of the endometrium, including proliferative endometrium (PEM), hyperplasia without atypia, AH, and well-differentiated endometrioid carcinoma. Slides were reviewed twice for diagnosis, with an intervening evaluation of a checklist of histologic features. Intraobserver and interobserver agreement were assessed using the kappa statistic. Intraobserver kappa values ranged from 0.67 to 0.89 (76% to 89% agreement). Interobserver kappa values by diagnostic category were: proliferative endometrium: 0.86; hyperplasia without atypia: 0.60; AH: 0.47; well-differentiated endometrioid carcinoma: 0.83; with a kappa value of 0.69 for all cases combined. Associations between the selected histologic features and the given diagnoses for each pathologist were analyzed using multiple logistic regressions to identify features that were useful for distinguishing among diagnostic categories. Histologic features determined by univariable and multivariable analyses that were found to be most associated with distinguishing diagnostic categories were: proliferative endometrium versus hyperplasia without atypia: gland crowding (univariable, multivariable), and gland branching (univariable); hyperplasia without atypia versus AH: presence of nucleoli (univariable, multivariable), nuclear enlargement (univariable), vesicular chromatin change (univariable), nuclear pleomorphism (univariable), chromatin irregularities (univariable), and loss of polarity (univariable); hyperplasia without atypia versus carcinoma: glandular confluence/complex cribriform pattern (univariable, multivariable), stromal alteration (univariable, multivariable), and necrosis (univariable). In summary, interobserver agreement was good but was lowest for AH. Only the presence of nucleoli was strongly associated with distinction of AH from hyperplasia without atypia. Individual pathologists use additional features to diagnose atypia, but these features are not consistently associated with that diagnosis. Cribriform architectural pattern and stromal alteration were associated with the distinction of well-differentiated endometrioid carcinoma from AH.

p53 mutations and clonality in vulvar carcinomas and squamous hyperplasias: evidence suggesting that squamous hyperplasias do not serve as direct precursors of human papillomavirus-negative vulvar carcinomas.

Previous studies of vulvar carcinomas have shown two distinct subsets with respect to several clinicopathologic features. In younger women, the tumors are frequently human papillomavirus (HPV) positive, are usually of basaloid or warty histology, and are associated with vulvar intraepithelial neoplasia. In older women, the tumors are usually HPV negative, are typical keratinizing squamous carcinomas, and are associated with squamous hyperplasia--a lesion that has been purported to serve as a precursor to HPV-negative invasive carcinoma. In squamous carcinomas of the cervix, p53 inactivation (through gene mutation or interaction with the HPV E6 oncoprotein) occurs in most cases. Comparatively few studies have assessed p53 mutation and HPV status in vulvar carcinomas, and none has used molecular markers to evaluate squamous hyperplasias as direct precursors of HPV-negative invasive cancers. Of 18 invasive squamous carcinomas analyzed, seven (39%) were found to be HPV positive. Four p53 gene mutations were identified--all in HPV-negative tumors. DNA was subsequently prepared from microdissected archival tissues from all four specimens showing p53 gene mutations. DNA was separately isolated from normal squamous epithelium, invasive squamous carcinoma, and associated squamous hyperplasia. In each specimen, the p53 mutation was confirmed in the invasive tumor and absent in both normal and hyperplastic epithelium. To further investigate squamous hyperplasia as a potential precursor of HPV-negative invasive carcinoma, the authors determined the clonality of hyperplastic lesions adjacent to invasive carcinomas with p53 mutation. Clonality analyses were performed using a polymerase chain reaction (PCR)-based assay for X chromosome inactivation. Although all three informative carcinomas tested were monoclonal, corresponding normal epithelia and hyperplastic lesions were polyclonal. These findings underscore the heterogeneity of vulvar cancers with respect to loss of wild type p53 function either by interaction with the HPV E6 oncoprotein or somatic mutation of p53, and suggest that squamous hyperplasias do not serve as direct precursors of HPV-negative squamous carcinomas.

Chromosome abnormalities in primary endometrioid ovarian carcinoma.

Specific and recurrent chromosome abnormalities may occur in regions of the genome that are involved in the conversion of normal cells to those with tumorigenic potential. Ovarian cancer is the primary cause of death among patients with gynecologic malignancies. We performed cytogenetic analysis in a subgroup of epithelial ovarian tumors, the endometrioid tumors, which are histologically indistinguishable from endometrial carcinoma of the uterus. We studied 10 endometrioid tumors to determine the degree of cytogenetic similarity between these two carcinomas. Six of 10 endometrioid tumors showed a near-triploid modal number, and one had a tetraploid modal number. Eight of the 10 contained structural chromosome abnormalities, of which the most frequent were 1p-- (5 tumors), 6q-- (4 tumors), 19q+ (4 tumors), and chromosome 3 rearrangements (4 tumors). These cytogenetic results resemble those reported for papillary ovarian tumors and differ from those of endometrial carcinoma of the uterus. We conclude that despite the histologic similarities between the endometrioid and endometrial carcinomas, the genetic abnormalities in the genesis of these tumors differ significantly.

Effect of tryptophan on growth and morphology of Hansenula schneggii cells.

Sundhagul, Malee (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Effect of tryptophan on growth and morphology of Hansenula schneggii cells. J. Bacteriol. 92:241-249. 1966.-When Hansenula schneggii cells were cultured aerobically in a tryptophan-glucose medium, 70 to 90% of the cells were elongated; no growth occurred under anaerobic conditions. The size of the elongated cells was 15 to 25 mu by 2 to 4 mu, as compared with 2.5 to 5 mu for ellipsoidal cells. Formation of elongated cells occurred essentially during the logarithmic growth period; the highest percentage of elongated cells was found soon after the end of this growth phase. In the later stationary phase, some of the cells formed spherical buds which became spherical cells. The rate of cell division during this period was greatly reduced, but the spherical cells formed decreased the percentage of elongated cells in the population. Cells cultured in a membrane-filter filtrate of a tryptophan-glucose medium (with limiting tryptophan), in which elongated cells had been grown, were ellipsoidal until nitrogenous nutrients were exhausted; thereafter the cells were elongated if tryptophan was added. Of compounds related to tryptophan, kynurenine was the only one which induced a high percentage of the cells to elongate. Some amino acids, such as cystine, histidine, phenylalanine, tyrosine, and threonine, induced elongation in about 15% of the cells. Growth of cells with other amino acids, or the addition of most of the other amino acids to tryptophan-glucose medium, resulted in a population of spherical cells. Several consecutive sequential transfers of cells into tryptophan medium induced elongation in 90% of the cells, but one transfer from a culture with elongated cells into a medium with ammonium sulfate, or a mixture of amino acids, gave a culture with ellipsoidal cells. Growth in media at pH 4 or 5 favored formation of elongated cells; as the pH was increased, the percentage of elongated cells decreased. Carbon sources other than glucose did not affect the percentage of elongated cells, except for the alcohols mannitol and erythitol, which gave comparable growth but reduced the percentage of elongated cells from 70 to 50%. Cell wall analyses of the two types of cells indicated that elongated cells have 2.5 times as much mannan as cell walls of ellipsoidal cells. This suggests that tryptophan, kynurenine, and, to a limited extent, some of the other amino acids cause a diversion of polysaccharide biosynthesis to mannan in the elongated cells rather than to glucan as in ellipsoidal cells.