New Pathways In Cancer Research

8/3/2019 New Pathways In Cancer Research

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UNIVERSIDAD COMPLUTENSE DE MADRID,

(Spain)

FACULTAD DE MEDICINAFACULTAD DE MEDICINA

- new relevant biomolecular concept, its

applications in cancer research andother research fields

A. Ferreira-Alemo, MD PhD

RIBOGRAMA:

New Pathways inCancer Research


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The purpose of this research, which was presented as a Doctoral Thesis, at

UNIVERSIDAD COMPLUTENSE DE MADRID is draft a method of:

- diagnosis,

- screening and

- monitoring

of the colon and rectum cancer, founded on technical and current concepts of Molecular Biology

This new method allows a diagnosis in a "pre-carcinoma in situ stage,

since it is based on the phenotype of mucosal cells, composed ofvery high

quantities of free ribosomes in the cytoplasm of colonocytes, which can be

quantified by means of the of the flow cytometry, after its isolation, and

labelled with fluorochromes.

The repeated records of those quantities of free ribosomes, establishes a

graphic curve (RIBOGRAMA) that represents the degree of a malignanttendency, which above a certain level of concentration, allows to say that the

cells in a tissue (of the colon and rectum, e.g.) can be considered in the process

of developing carcinoma in the colon-rectum mucosa, before any macroscopic

viewing (endoscopy).


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biomolecular basis of the cellular machinery

FREE AND MEMBRANE RIBOSOMES

Free ribosomes are generally assumed to synthesize intracellularproteins (internal

economy of the cell), whereas bound ribosomes synthesize proteins intended for

secretion (exportation).

DNA

RNA

CELLULARNUCLEUS CYTOPLASM

ROUGHENDOPLASMICRETICULUM

FREERIBOSOMES

MEMBRANE

RIBOSOMES


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Schematic three-dimensional rough endoplasmic reticulum, showing the synthesis of

proteins destined for export by ribosomes attached to membranes reticulum

(signal sequence).


MEMBRANE RIBOSOMES


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RIBOSOME

TumorMarkers

U p s t r e a m s i d e Downstream side

The protein synthetic capacity of a cell is

dictated by a number ofprocesses such:


CONTROL OF PROTEIN SYNTHESIS

mRNA availability; efficiency of translation; availability of translation factors;

number of ribosomes


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THE RIBOSOME AS THE COMMON ELEMENT IN THE

CELLULAR BIOMOLECULAR PROCESS

1. The evidence accumulated to date

indicates that overall the protein syntheticcapacity is determined by the number of

free ribosomes, which function as an

informative sensorof the protein synthesis.

2. Increased cell proliferation is a hallmark

of agressive malignant neoplasms, whichrequires a general increase in protein

synthesis and a specific increase in the

synthesis of replication-promoting proteins.

3. Ribosome synthesis, or biogenesis, is a complex process dependent on the

coordinated synthesis of approximately 85 ribosomal proteins, four ribosomal RNA(rRNA) and their subsequent processing and assembly into mature ribosomes.

(Tumor Markers)


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It has been shown in polyoma virus-transformed cells that one of the key mechanisms

for the loss of control ofprotein synthesis is an inability to decrease ribosome number,

which correlated with proliferation of transformed cells in fresh media without addition

of serum growth factors (Stanners et al., 1979).

CELL CYCLE

Hours Hours

Minutes

FREERIBOSOMES

QUANTITY

INTERPHASE MITOSIS INTERPHASE MITOSIS

G1 S G2 M G1 S G2 M

9,3 8,0 2,0 0,7 9,3 8,0 2,0 0,7

PRO MET ANA TEL

25,2 2,1 2,1 12,6

NORMAL CELL

NORMAL CELL

HYPERPLASIACELL

HYPERPLASIACELL

MALIGNANTTRANSFORMED

CELL


THE RIBOSOME AS THE COMMON ELEMENT IN THE

CELLULAR BIOMOLECULAR PROCESS


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RIBOGRAM CONCEPT

- From the subjective nature, relatively of the amount offree ribosomes,

a diagnosis of malignancy is made just under the idea of the constancy

property that consists of an great increase offree ribosomes in cells that

are in a process of multiplication without self control, as in the case ofmalignant cells;

- A malignant cell has a very high free ribosomes density that is a

fundamental aspect of its phenotype;

- So, naturally, to describe the quantities of free ribosomes it is

necessary to create a language reproducible, not subjective, with

mathematical translation, through a graphic curve that we callRIBOGRAMA.


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A perspective on the biology of malignant cells

Several lines of evidence, some well

established and recent, sayemphatically the idea that RNA

content is high in cancer cells and

genetic events that lead to cancer

are often linked directly or indirectly

to the ribosome biogenesis.


RIBOGRAM CONCEPT


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RIBOGRAMAGRAPHICCURVE

Legend:

smt = every 6 months; A, B, C, D =Concentration levels of free ribosomes


RIBOGRAM CONCEPT


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In the decades of 60s and 70s were

published studies concerning the

accumulation of free ribosomes duringchemical induction of neoplastic growth,

through initiation by 7,12-

dimethylbenz(a)anthracene and promotion

caused by 12-O-tetradecanoyl-phorbol-13-

acetate, in the interfollicular areas of the

dorsal skin of mice.

Accumulation of ribosomes in the process of oncogenesis


RIBOGRAM CONCEPT


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Case Number Quantitative description of free

ribosomes in EM (Electrom Microscope)

Electrom Microscope Main General

View

Diagnosis / Information References

(Publishedi n the Doctoral Thesis)

1 Numerous free ribosomes Citoplasma filled numerous

organelos, mitochondrias, free

ribosomes, short

Carcinoma pappilar invasivo,

moderadamente diferenciado

396

2 Free ribosomes randomly dispersed Small clusters of free ribosomes Glial C6 Cells transformadas 397

3 Free ribosomes excedingly abundant --------------------- Human adrenocortical carcinoma

derived SW-13 cell lines

398

4 Numerous free ribosomes Few granular endoplasmic reticulum Human osteosarcoma 399

5 Increased free ribosomes Membrane bound ribosomes

decreased

Liver tumor rats 400

6 Free ribosomes Few organelles Hepatoma cell lines 401

7 Numerous free ribosomes and

polysomes

Some rough endoplasmatic

reticulum

EBV Related liver tumor (occurring

after kidney transplant)

402

8 Increase of free ribosome numbers --------------------- HEL cells human erytroleukemiainduced

403

9 Principal cytoplasmic organelles were

free ribosomes into rosettes

Membranes of rough endoplasmic

reticulum and Golgi apparatus were

poorly developed in most neoplastic

cell

Bronchogenic carcinoma 404

10 Rich in ...free ribosomes Rich in organelles of this type

including microtubules

mitochondria

Oligodendroglioma 405


TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION

(FIRST 63 CASES AMONG A TOTAL OF 205)


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Case

Number

Quantitative description of free


Electrom Microscope Main General View Diagnosis / Information References

(Publishedi n the Doctoral Thesis)

19 exhibited greater activity (free

ribosomes)

Hepatoma transplantable 414

20 Turnover of free ribosomes was

greater in host livers

Transplantable hepatoma rat cells 415

21 Free ribosomes were the most prominent

cellular organelles

Moderate number of mitochondria, were

randomly distributed. Poorly developed

granular endoplasmatic reticulum

Nasofaringeal carcinoma 416

22 Disagregation of free and membrane

bound polyribosomes

---------------------- Effect of aflatoxin B1 on hepatic

polyribosomes and protein sintesis

in the rat

417

23 Free ribosomes Few organelles, small number of

mitochondria, poor developed Golgi

complexes inconspicuous rough

endoplasmic reticulum

Keratin positive Ewings sarcoma 418

24 Prominent ultrastructural features

being free ribosomes

Prominent

- rough endoplasmic reticulum cisternal -

mitochondria

Human adrenocortical carcinoma

derived SW-13 cell line

419

25 Prominent free ribosomes Prominent rough endoplasmic reticulum Thalamic tumor in a patient with

human T-cell lymphotropic virus

type 1 (HTLV-1) [extranodal

lymphoma of the skull]

420





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Case

Number




View


(Published in the Doctoral Thesis)

11 Numerous free ribosomes Enlarged mitochondria prominente

smooth and rough endoplasmic

reticulum

Mammary Paget Disease 406

12 Numerous free ribosomes ----------------------- Intestinal tumor induced by 1,2 -

dimethylhydrazine

407

13 Increase in membrane free ribosomes Functional alterations paralleled by

an increase in membrane free

ribosomes

Rat liver hepatocarcinogenesis

induced by 0,25% DL ethiomine

[hepatoma]

408

14 cytoplasmicmainly on free ribosomes Weakly rough surface endoplasmic

reticula cisternae and Golgi

complexes

Neuroblastoma cell lines 409

15 After 2 to 3 weeks ofATRA treatment

ER and free ribosomes became rare as

the maturation process

Acute promielocytic leukaemia cells 410

16 Cytoplasm may contain free ribosomes Few mitochondria, a dilated rough

endoplasmic reticulumMetastatic (neck lymph node)malignant extrarenal rhabdoid tumor

411

17 predominantly free ribosomes Inconspicuous rough

-endoplasmic reticulum

-small number of mitochondria

-developed Golgi complexes

Ewings sarcoma 412

18 Free ribosomes w ere present Mitochondria endoplasmic

reticulum and Golgi apparatus were

present

Colorectal polyps 413





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Case

Number

Quantitative description of free ribosomes

in EM (Electrom Microscope)


View



26 Accumulation of numerous free ribosomes Marcadly increased vesicular

trafficking the Golgi

Neuroblastoma B-104 cells 421

27 Free ribosomes Cytoplasmic organelles such as

rough endoplasmic reticulum,

mitochondria

Carcinoma of the thyroid gland 422

28 Numerous free ribosomes Abundance of dilated rough


Small cell carcinoma of the ovary 423

29 Abundant free ribosomes -Absent smooth endoplasmic

reticulum

Carcinoma of the endometrium 424

30 Abundant free ribosomes Abundant rough endoplasmic

reticulum cisternae

Smooth endoplasmic reticulumabsent

Adrenocortical oncocytoma 425

31 Abundant free ribosomes Prominent nucleoli Chemically induced mouse

hepatoblastoma

426

32 Numerous free ribosomes Small amounts of rough

endoplasmic reticulum and round

mitochondria

Malignant fibrous histiocytoma 427

33 Numerous (?) free ribosomes Numerous small mitochondria Adenocarcinoma cells of an ovarian

clear cell tumor

428





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Case

Number




View



34 Increase of free ribosomes Prominent nucleoli, marked decrease

in rough endoplasmic reticulum;

abundant microphilaments and

microtubules

Histogenesis ofpseudoductular

changes in pancreas of guinea pigs

with N-methyl, N-Nitrosourea

429

35 Many free ribosomes Large nuclei with prominent nucleoli;

small quantities of rough and smooth


NCI H295R cells 430

36 Free ribosomes were common -Rich mitochondria

- Some rough endoplasmic reticulum

Leiomyoblastoma of the uterus 431

37 Increased free ribosomes Membrane bound ribosomes

decreased

Liver tumor rats 432

38 High content s o f free ribosomes ----------------------- Sarcoma 45 433

39 Clusters of free ribosomes Dilated endoplasmic reticulum

profiles

Cortical dendritic spines 434

40 Many free ribosomes Many rough endoplasmic reticuli Pulmonary clear cell carcinoma 435

41 Numerous free ribosomes ----------------------- Hepatocarcinogenesis induced by

ethionine

436





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Case

Number




View



42 ...Contain a few cytoplasmic organelles

...except for free ribosomes

Contain few cytoplasmic organelles

and a small number of

mitochondria

Human germ cell tumor derived cell

line

437

43 tumor cells contains free ribosomes

contain filamentous structures Embryonal rhabdomyosarcoma in bucal

mucosa

438

44 Abundant free ribosomes Abundant mitochondria Oncocytic metaplasia and carcinoma of

the endometrium

439

45 referncia a free r ibosomes ...Referncia a

-Golgi complex

-Mithocondria

-RER

Tapioca melanoma of the iris... 440

46 free ribosomes w ere present Rough endoplasmic reticulum were

present

lipomatous lesions in the liver of

B6C3F1 mice

441

47 numerous free ribosomes numerous distended rough

endoplasmic reticula, Golgi

apparatus

Pituitary fibrosarcoma following

radiotherapy

442

48 Abundant free ribosomes Abundant mitochondria, lysosomes,

rough surface endoplasmic

reticulum

Epithelioid malignant schwannoma cell

line

443



(FIRST 63 CASES AMONG A TOTAL OF 205))


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Case

Number




View

Diagnosis / InformationReferences


49 free ribosomes were observed desmosomes present Rat bladder tumor cell lines 444

50 increases of free ribosomes Nuclear lobulation and nucleolar

content

Human erythroleukemia cells 445

51 Numerous free ribosomes Some mithocondria and other

poorly developed cytoplasmic

organelles, suggesting

undifferentiated nature

Rat malignant fibrous histiocytoma 446

52 Many free ribosomes ----------------------- Rat colorectal epithelium tumors after

treatment with carcinogen

447

53 Numerous free ribosomes Poorly differentiated cytoplasm

numerous mitochondria

Small cell osteosarcoma 448

54 Abundance of free ribosomes Paucity of organelles Ewings sarcoma 449

55 Numerous free ribosomes Little rough endoplasmic

reticulum; few mitochondria; small

Golgi complex

Mouse mammary gland

adenocarcinomas

450

56 Great number of free ribosomes ----------------------- Malignant lymphoma 451





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Case

Number

Quantitative description of free ribosomes in

EM (Electrom Microscope)

Electrom Microscope Main General View Diagnosis / InformationReferences


57 Large amount of free ribosomes Well developed Golgi apparatus, rough

endoplasmic reticula

Monocytic leukaemia cells 452

58 Free ribosomes Roughendoplasmic reticulum

pleomorphic mitochondria

Poorly differentiated Rhabdomyosarcomas 453

59 805 free ribosomes ----------------------- Lymphocytic leukaemia tumor 454

60 Clusters of free ribosomes ProminentGolgi apparatus numerous

profiles of rough endoplasmic reticulum

Adenocarcinoma from apocrine glands in dogs 455

61 Few free ribosomes Poor developed Golgi apparatus

phenotypic features characteristics for

myeloma cells

Myeloma cells in human hybridoma 456

62 Numerous free ribosomes Few granular endoplasmic reticulum Human osteosarcoma 457

63 Many free ribosomes Numerous organelles many

lysosomes, swollen mithocondria, Golgi

complexes, rough and smooth


Subependymal giant cell tumor 458





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TABLE II - DIFFERENT WAYS HOW IS DESCRIBED THE CONTENT OF FREE RIBOSOMES IN

ATYPICAL CELLS, ON E.M. - ELECTRON MICROSCOPE (example of the ten cases of table i)

N de caso Descripcin cuantitativa de los ribosomas

libres

1 Numerous free ribosomes

2 Free ribosomes randomly dispersed

3 Free ribosomes excedingly abundant4 Numerous free ribosomes

5 Increased free ribosomes

6 Free ribosomes

7 Numerous free ribosomes and polysomes

8 Increase of free ribosome numbers

9 Principal cytoplasmic organelles were freeribosomes into rosettes

10 Rich in ...free ribosomes

Biomolecular analysis of malignant cells

Content of free ribosomes in malignant cells


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TABLE III-A - (NUMEROUS FREE RIBOSOMES)

Case

NumberDiagnosis/Information

Case


1 Papilar Invasive Carcinoma 50 Small cell osteosarcoma

9 Mammary Paget disease 52Mouse mammary gland

adenocarcinoma

10 Intestinal Tumor 59 Human osteosarcoma

24 Neuroblastoma 75 Testicular seminoma

26 Small cell carcinoma of the ovary 87 Phylloides tumor of the prostate

30 Malignant fibrous histiocytoma 88 Malignant Choroid Plexus papiloma

31 Adenocarcinoma of ovary clear cell 91 Malignant fibrous histiocytoma

38Hepatocarcinogenesis induced by

ethionine105 Small cell carcinoma of ovary

44 Pituitary fibrosarcoma 107 Mammary Paget disease

48 Rat malignant fibrous histiocytoma 108 Adenocarcinoma of stomach


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Case


Case


117 Small cell carcinoma of the ovary 142 Testicular seminoma

118 Neuroblastoma 151Mouse mamary gland

adenocarcinoma

123Rat hepatocarcinogenesis induced

by ethionine153 Osteosarcoma

125 Small cell osteosarcoma 163 Mt TW15 mamosotrofic tumor

127Transplantable Rat malignant

fibrous histiocytoma178

Hyperplasic foci in precancerous

rat liver

129 Pituitary adenoma 191 Human tongue carcinoma cells

131 Large malignant choroid plexus 193 Intestinal tumors

132 Control neurocitoma

LEGEND:Sample of cases 37Phenotypes of malignancy observed 35

PERCENTAGE OF MALIGNANT TISSUES - 95%


TABLE III-A - (NUMEROUS FREE RIBOSOMES)


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TABLE III-B - (RICH IN FREE RIBOSOMES)

Case Number Diagnosis/ Information

8 Oligodendroglioma

96 Adenocarcinoma of the stomach

106 Oligodendroglioma

140 Naked nuclei in breast aspirate smears

197 Epidermal carcinogenesis


PERCENTAGE OF MALIGNANT TISSUES - 100%


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TABLE III-C - (ABUNDANT FREE RIBOSOMES)

Case


Case


3 Adrenocortical carcinoma 90Chemically induced mouse

hepatoblastoma

27 Carcinoma of the endometrium 95 Adrenocortical carcinoma

28 Adrenocortical oncocytoma 98 Carcinoma of thyroid gland

29Chemical induced mouse

hepatoblastoma104 Ductal papillary adenocarcinoma

41 Carcinoma do endometrium 115 Carcinoma of endometrium

45

Epithelial malignant schwannoma

cell line 116 Adrenocortical oncocytoma

51 Ewings sarcoma 128Epithelioid malignant schwannoma

cell lise

69 Brain tumor in doges 138 Mouse hepatoblastoma

71 Vaginal hemangiopericytoma 145 Vaginal hemangiopericytoma

80 Mouse hepatoblastoma 146 Brain tumor


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Case


Case


156 Extra skeletal Ewings sarcoma 176 Adrenal cortical tumor

160 Neuroblastoma 183 Pseudolymphoma of the lung

165 Epithelial tumors induced in ratkidney

187 Salivary gland carcinoma

166 Hepatoblastoma of foetal liver 188 Epithelioid sarcoma

168 Hepatocellular tumors 189Malignant epithelioid

schwannoma

171 Stromal sarcoma of breast 199Primary lymphosarcoma of

testis

173 Cholangiocarcinoma 201 Ependymoma


PERCENTAGE OF MALIGNANT TISSUES - 94,4%


TABLE III-C - (ABUNDANT FREE RIBOSOMES)


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TABLE III-D - (MANY FREE RIBOSOMES)

Case


Case


33 NCI-H295R cells 124 Pulmonary clear all carcinoma

37 Pulmonary clear cell carcinoma 126 Colorectal tumors

49 Colorectal epithelium tumors130 Human neuroblastoma cell line

60 Subependymal giant cell tumor 137 Malignant fibrous histiocytoma

73 Pancreatic islet cell tumor 143 Pancreatic islet cell tumor

89 Neuroblastoma cell line 155 Malignant lymphoma

101 Primary osteoblast like cells 164 Spontaneous testicular neoplasm

102 Human pituitary tumor 196

Epidermis experimental

carcinogenesis

112NCI-H295R cells (human adrenal

cell lines)202 Malignant lymphoma

122Pluripotent human germ cell tumor

derivated cell lineLEGEND:Sample of cases 19Phenotypes of malignancy observed 17



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TABLE III-E - (INCREASED NUMBER OF FREE RIBOSOMES)

Case


Case


6 Human erythroleukemia cells 62 Papillary thyroid carcinoma

11 Rat liver hepatocarcinoma induced 77 Medullary carcinoma of the thyroid

15 Ewings sarcoma 79 Cutaneous neurofibromas

19 Nasofaringeal carcinoma 84 R and T head and neck carcinomas

22 Adrenocortical carcinoma 100 Human erythroleukemia cells

23 Thalamic tumor 109 Transplantable hepatomas

32Pseudoductular changes in pancreas

pigs110

Adrenocortical carcinoma

34 Leiomyoblastoma of the uteros 113Pseudo-ductular changes in

pancreas of pigs

35 Liver tumor rats 120 Embryonal Rhabdomyosarcoma

47 Human erythroleukemia cells 133Seminoma and parathyroid

adenoma


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Case


Case


141 Medullary carcinoma 181Malignant fibrous histiocytoma of

the orbit

157 Oesophageal epithelium dysplastic

foci of the thyroid185 Clear cell thyroid carcinoma

159 Pituitary adenomas 186 Hepatocarcinoma

167 Uveal malignant melanoma 204Carcinogenesis induced by 4-

dimethylaminobenzene




TABLE III-E - (INCREASED NUMBER OF FREE RIBOSOMES)


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TABLE III-F - (FREE RIBOSOMES)

Case


Case


2 Glial C6 cells transformed 39Human germ cell tumor derived cell

line

5Administration of ethionine female

rats40 Embryonal rhabdomyosarcoma

7 Bronchogenic carcinoma 42 Tapioca melanoma of the iris

12 Neuroblastoma cell lines 43Lipomatous lesions in the liver of

B6 C3 F1 mice

14Malignant extra renal rhabdoid

tumor46 Rat bladder tumor cell lines

16 Colorectal polyps 55 Poorly differentiated

rhabdomyosarcoma

17 Hepatoma transplantable 56 Lymphocytic leukaemia tumor

21 Ewings sarcoma 57Adenocarcinoma from aprocrine

glands in dogs

25 Carcinoma of thyroid gland 63 Carcinoma of lung

36 Cortical dendritic spines 64 Friend erythroleukemia cell growth


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Case


Case


65 Hodgkins disease 82 Rat pheochromocytoma cell

66 Hepatoma cells 83 Carcinoma of endometrium

67 Hepatoma cells 85 Myelogenous leukaemia cell line

68 Astroblastoma 86Seminoma and parathyroid

adenoma

70 Endometrial adenocarcinoma 92 Myeloma cells

72 Clear odontogenic tumor 94 Congenital fibrosarcoma

74 Cell condrosarcoma 97 Human tumoral ependymal celllines

76 Sphenoidol ridge meningioma 99Ductal adenocarcinoma of the

pancreas

78 Osteosarcoma cell line 103 Carcinoma rat testis

81 Malignant fibrous histiocytoma 111 Human adrenal cell lines




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Case


Case


119 Cerebral small cell tumor 150 Carcinoma of the lung

120 Embryonic rhabdomyosarcoma 154Adenocarcinomas (in dogs),

aprocrine glands

121 Nevic corpuscle 158 Branchiometric paragangliomes

134 Myelogenous leukaemia cell line 161 Adrenocortical carcinomas

135 Carcinoma of the endometrium 162 Melanocytomas of the optic nerve

136Adrenal chromaffin and

pheochromocytoma169 Transitional cloagenic carcinomas

139

N-methyl-N-amilnitrosamina

induced rat oesophageal

carcinogenesis

170 Maxilofacial synovial sarcoma

144 Clear odontogenic tumor 174 Cultured hepatoma cells

148 Rat ascitis hepatoma cells 175 Monoclonal Hodgkin cells cultured

149 Rat ascitis hepatoma cells 177 Gastric leiomyosarcoma




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Case Number Diagnosis/Information

180Establishment of a human rectal cancer

cell line

182 Pituitary adenomas

184 Myeloma cells

195Adenocarcinoma of the human tuba

uterina

198 Pituitary thyrotrophic tumor

200 Dermatofibroma (histiocytoma)

205 Friend erythroleukemia cell growth

LEGEND:Sample of cases- 70Phenotypes of malignancy observed 67





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Table of

"groups of expressions"

Number of cases

in each group of

expressions

Malignant phenotypes in

each "group of

expressions"

%Malignant

tissues

I - (NUMEROUS FREE RIBOSOMES) 37 35 95 %

II (RICH IN FREE RIBOSOMES) 5 5 100 %

III (ABUNDANT OF FREE RIBOSOMES) 36 34 94,4 %

IV (MANY FREE RIBOSOMES) 19 17 89,4 %

V (INCREASED NUMBER OF FREE

RIBOSOMES)38 35 92,2 %

VI (FREE RIBOSOMES) 70 67 95,7 %


TABLE OF "GROUPS OF EXPRESSIONS" RELATIVELY

TO DESCRIBE THE CONTENT OF FREE RIBOSOMES


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THE RIBOSOME NUMBER AS A QUANTITATIVE PARAMETER

OF THE MALIGNANT PHENOTYPE TREND

1. The RIBOGRAME graphic curve reflects a dynamic idea of

changes in numerical values or quantities of all the free ribosomes.

2. The density offree ribosomes is the trend of the phenotype of the

malignancy of a cell, relatively to a normal range, of a community of

cells under observation in regard to biomolecular behavior,

representing a malignant growth of the cell phenotype, above a

certain level of increase.

3. It will be very important produce predictive mechanistic models

based on tumor dynamics, reflecting the translation in the cell

phenotype, for example (the count of free ribosomes) in a cell as

the phenotypic alteration process under the oncogenic stimulus.


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THEORETICAL CONSIDERATIONS ABOUT THE GRAPH

CURVE OF RIBOGRAMA AND ITS IMPLICATIONS IN THE

CLINICAL PRACTICE AND OTHER RESEARCH FIELDS

Legend:

smt = every 6 months; A, B, C, D =Concentration levels of free ribosomes


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INTERPRETATION OF THE GRAPH IC CURVE (RIBOGRAMA)

Curve that starts at the normal level and reaching the increased level,

which involves care for a prophylactic study on the reasons or causes that determine itsappearance. The cells at this level (increased) still have no security features of malignancy, but

their increased number of ribosomes continues growing. The cell is transformed into a

malignant phenotype (alarm level). At this point it is essential to make a dietary study to survey

and identify possible mutations responsible for the uncontrolled growth of ribosome biogenesis

in the process of carcinogenesis;

Segment of the curve derived from the anterior (segment a-b), which

already corresponds to the phenotypic alterations seen in electron microscopy, in which cells

are already present with phenotypic features of malignancy (significant increase in median

density of free ribosomes), which is the alarm level


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Segment of the curve, continuing the anterior segment, which represents the level of

signs and symptoms of the CRC disease (clinical level);

This segment of the curve corresponds to a change in direction of the curve a-b that

results from preventive action and treatment (medications and special diet)

Corresponds to a curve in which cells grow (multiply or proliferate) in a self-proliferation

mechanisms increased, as in the case of the seminiferous tubules, or the endometrium. This increased level of

ribosome biogenesis control is thereof within the physiological (non-malignant);

Normal curve

This work is intended to study the item colorectal cancer (CRC), in which ribogramacurve is high, so it is not the time to talk about the meaning of it at low level. Still, in this level, it will be useful tostudy conditions of degeneration and involution of cells and tissues, either the intestinal mucosa, or other extra-

intestinal tissue.


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CHARACTERISTICS OF MALIGNANCY VERSUS THE

COUNTING OF FREE RIBOSOMES

While Ribograma method gives a quantity idea of the cell phenotipic view, the descriptions

of Electron Microscope (EM) view of the cells with phenotypic characteristics of malignancy vary

by greater or lesser degree of differentiation of its texture or morphology, but these descriptions are

subjective, depending on the observer.

In terms of molecular biology of cancer, the malignant transformation of the normal cells is the

acquisition of a series of progressive specific genetic changes that act disobeying to the strong

antitumor mechanisms that exist in all normal cells, which include: a) - regulation of signal

transduction, b) - differentiation, c) - apoptosis, d) - DNA repair; e) - cell cycle progression, f) -

angiogenesis, g) - cell adhesion, which are not quantifiable, and in its whole do not allow a

monitorization reproducible and practical .

As in the descriptions of EM, these mechanisms of malignant transformation cannot be evaluated

by mathematic criteria, because the described features cannot be quantified. Similarly, the properties

of malignant transformed cells, growing in cell culture or in vivo, as observed by techniques of cell

biology or molecular biology do not allow a rigorous quantitative assessment, because they

depend on subjective criteria, without concrete numbers or standardized numerical limits.


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CHARACTERISTICS OF MALIGNANCY VERSUS THE

COUNTING OF FREE RIBOSOMES

A. The characteristics listed in next table are properties of malignant transformed cells

growing in cell culture (or in vivo), that are not susceptible of being quantified, while with

the quantification free ribosomes it is possible to make comparisons, through counting

them with the use offlow cytometry techniques. Then, with the count of free ribosomes ispossible to compare the results with reference to the time variable, like it is done with theRIBOGRAMA concept.

B. Of the characteristics listed in the next table it is not possible to register a numeric result,

because they do not show a mathematic and reproducible dynamic trend growth andproliferation status of a set of cells belonging to a given tissue, but

C. This is possible with the RIBOGRAMA concept.


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PROPERTIES OF GROWING MALIGNANT TRANSFORMED

CELLS IN A CULTURE OF CELLS AND /OR IN VIVO

1. Cytological changes similar to cancer cells in vivo, including increased cytoplasmic basophilia, number and size of the nuclei increased, increased nucleus-

cytoplasm relation, formation of clusters and cords of cells;

2. Altered growth characteristics;

a. "Immortality" of the transformed cells in culture. Transformed malignant cells become "immortal" in that they can be transferred in culture indefinitely;

b. Decreased dependent inhibition of cell density or loss of "contact inhibition ." The transformed cells often grow to a density higher than their normal

counterparts, and they can pile up in culture rather than stop growing when they contact each other;

c. Decreased serum needs. The transformed cells require decreased concentrations of serum or growth factors to replicate in culture compared to cells not

transformed.

d. Loss of anchorage dependence and acquisition of the ability to grow in soft agar. The transformed cells may lose their need to grow attached to surfaces and

can grow as colonies in semisolid free.

e.Loss of cell cycle control

. The transformed cells do not stop in G

1, or borderline G

1 /S cell cycle when subjected to metabolic constraints of growth

.

f. Resistance to apoptosis (programmed cell death).

3. Changes in the structure and function of the cell membrane - including increased agglutination by plant lectins, altered composition of cell surface glycoproteins,

proteoglycans, glycolipids and mucins; appearance of tumor-associated antigens, and increased uptake of amino acids, hexoses and nucleotides.

4. Loss of cell-cell and cell-extracellular matrix that promotes cell differentiation.

5. Loss of response to inducers of differentiation and altered cellular receptors for these agents .


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PROPERTIES OF GROWING MALIGNANT TRANSFORMED

CELLS IN A CULTURE OF CELLS AND /OR IN VIVO

6. Alteration of signal transduction mechanisms, including growth receptors constitutive phosphorylation cascades and mechanisms of dephosphorylation rather

than a regulated function.

7. Increased expression of oncogenic proteins due to translocation, amplification and chromosome mutation.

8. Loss of protein products of tumor suppressor genes due to deletion or mutation .

9. Genomic misreading that causes the overproduction of growth-promoting substances, eg, IGF-2.

10. Increase, or unruly production, of growth factors, e g, TGF-alpha, tumor angiogenesis factor, PDGF, hematopoietic growth factors (e g, CSFs, interleukins).

11. Genetic instability, leading to progressive loss of regulated cell proliferation, enhanced invasion and increased metastatic potential . Genes "mutator" may be

involved in this effect.

12. Altered enzyme profifes. The transformed cells have increased levels of enzymes involved in the synthesis of nucleic acids and produce higher levels of lytic

enzymes, e g, proteases, collagenases and glycosidases.

13. Production of oncodeveloping gene products. Many malignant transformed cells growing in culture or in vivo produce increased quantities of oncofetal antigen

(e g CEA), placental hormones (e g, chorionic gonadotropin), or isoenzymes feto-placental type (e g, placental alkaline phosphatase).

14. Ability to produce experimental animal tumors. This is the condition sine qua non that define the malignant transformation in vitro. I f the cells that are thought to

have been transformed do not product tumors in appropriate hosts animals, they can not be defined as "malignant". However, the failure to grow in an animal

model does not exclude the fact that they can be tumorigenic in a different kind of animal .

15. Ability to avoid host antitumor immune response.


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COLORECTAL CANCER

STATISTICS

In Europe colorectal cancer (cancer of the colon and rectum) is the

most common form of all the cancers.

Each year over 200,000 citizens in Europe die from colorectal

cancer (nearly two thirds of all cases diagnosed).

Colorectal cancer is the third most common cancer worldwide.

Yet this disease is preventable in most cases and highly treatable if

diagnosed in its very-early stage (pre-in situ stage ).


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COLORECTAL CANCER


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In colorectal cancer, to achieve the quantification of free

ribosomes there are, mainly, the following steps:

a) creation of a device (kit to collect stools which have thousands of mucosal cells

colonocytes - freed from the colonic and rectal mucosa, which are mixed with

and covering the stools voided);b) separation of the cells (colonocytes) from the stools;

c) homogenization and calibration of the colonocytes, through rupture of the

colonocytes, with certain routine techniques;d) isolation of the free ribosomes, though differential centrifugation;

e) labeling of the free ribosomes, utilizing fluorochromes (nanotechnology); and

f) counting labeled free ribosomes, utilizing flow cytometry or other method.

COLORECTAL CANCER (CRC)

PREPARATION AND COUNTING OF FREE

RIBOSOMES


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ADVANTAGES OF RIBOGRAMA OVER OTHER

NONINVASIVE METHODS OF DETECTECCION OF CRC

The degree of public support will be quite high, given sum of

the following nice factors ":

01- No invasive act;02- No need for the patient, to handle their own feces during collection;

03- No fecal odor nuisance since the fecal collector is industrially designed

accordingly (see the design of the collector of feces in the following

images);

04- No bowel preparation is needed;

05- No need for cleansing enemas;

06- It is not necessary to use cathartics;

07- It is not necessary to shift the patient to a medical appointment or to a

medical center;


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08 - The patient does not have to change their way of life;

09 - The patient does not have to change the routine of their daily activities or

work;

10 - The patient has no need to change the habits and type of food;11 - The patient does not have to stop, or modify any medication;

12The method does not give false negatives or false positives, because the

test specification is limited to counting the ribosomes of colonocytes,

which are the targets to be studied in cases of colorectal cancer, or in

cases under malignization process, meaning that the test is 100%

specific, 100% reliable and universal, for all tissues, once the cells tostudy are separated.


ADVANTAGES OF RIBOGRAMA OVER OTHER

NONINVASIVE METHODS OF DETECTECCION OF CRC


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RIBOGRAMA is an objective and quantitative method that provides an unique

service to HealthCare Providers worldwide. The method is qualified to deal within all

business sectors of the worldwide HealthCare, from the sole practice physician to the

largest chain of hospitals.

The advantages of RIBOGRAMA METHOD over other noninvasive tests of

cancer of the colon and rectum are the evident twelve "factors nice" that by themselves

provide a good patient compliance and a preference for determining the health

professionals, because there are no false positives or false negatives, since they areconsidering charges of ribosomes of cells isolated from the colorectal mucosa.

RIBOGRAMA

COLO RECTAL CANCER

FINAL REMARKS


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SUMMARYOF COLECTOR PARTS

SUPPORT PROTOTYPE OF STOOL COLLECTION

(COLLECTOR)


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(COLLECTOR)


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(COLLECTOR)


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(COLLECTOR)


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(COLLECTOR)


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The colorectal cancer represents one of the biggest incidences of human cancer in the western

type of life and cocked food.

This research project has strong potential in the following fields :

public health,

medico-hospitalar practice,

clinical and laboratorial research,

food industry,

veterinary,

oncologic research,

pharmaceutical industry and clinical trials research etc.,

The method has great medical and financial impact, firstly, by now, in the lowering significantly the

morbidity and mortality of the colorectal cancer disease, in the world, and, after, with other organs

and systems.

APLICATIONS


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Raise new questions, explore new possibilities, and

regard old problems from a new angle...

[Albert Einstein]


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The place where I was borned

and grown.

Oporto city - PORTUGAL

New Pathways In Cancer Research

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